Quaternary ammonium diphenylmethyl compounds useful as muscarinic receptor antagonists

ABSTRACT

The invention provides compounds of formula I: 
                         
in salt or zwitterionic form or a pharmaceutically acceptable salt thereof, wherein R 1-6 , a-e and Q are as defined in the specification. These compounds are muscarinic receptor antagonists. The invention also provides pharmaceutical compositions containing such compounds, processes for preparing such compounds and methods of using such compounds to, for example, treat pulmonary disorders such as chronic obstructive pulmonary disease and asthma.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. Ser. No.12/898,858, filed Oct. 6, 2010, now allowed; which is a divisionalapplication of U.S. Ser. No. 12/070,780, filed Feb. 21, 2008, now U.S.Pat. No. 7,834,185; which claims the benefit of U.S. ProvisionalApplication No. 60/903,110, filed on Feb. 23, 2007; the entiredisclosures of which are incorporated herein by reference in theirentirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to quaternary ammonium compounds havingmuscarinic receptor antagonist or anticholinergic activity. Theinvention also relates to pharmaceutical compositions comprising thesecompounds, processes for preparing them and methods of use to treatpulmonary disorders.

2. State of the Art

Pulmonary or respiratory disorders, such as chronic obstructivepulmonary disease (COPD) and asthma, afflict many millions of peopleworldwide and such disorders are a leading cause of morbidity andmortality.

Muscarinic receptor antagonists are known to provide bronchoprotectiveeffects and therefore, such compounds are useful for treatingrespiratory disorders, such as COPD and asthma. When used to treat suchdisorders, muscarinic receptor antagonists are typically administered byinhalation. However, even when administered by inhalation, a significantamount of the muscarinic receptor antagonist is often absorbed into thesystemic circulation resulting in systemic side effects, such as drymouth, mydriasis and cardiovascular side effects.

Additionally, many inhaled muscarinic receptor antagonists have arelatively short duration of action requiring that they be administeredseveral times per day. Such a multiple-daily dosing regime is not onlyinconvenient but also creates a significant risk of inadequate treatmentdue to patient non-compliance with the required frequent dosingschedule.

Accordingly, a need exists for new muscarinic receptor antagonists. Inparticular, a need exists for muscarinic receptor antagonists havinghigh potency, reduced systemic side effects when administered byinhalation, and a long duration of action thereby allowing foronce-daily or even once-weekly dosing. In addition, a need exists formuscarinic receptor antagonists having high affinity for the receptorand a long receptor half life. Such compounds are expected to beparticularly effective for treating pulmonary disorders, such as COPDand asthma, while reducing or eliminating side effects, such asdry-mouth and constipation.

SUMMARY OF THE INVENTION

The present invention provides novel quaternary ammonium compounds whichhave muscarinic receptor antagonist or anticholinergic activity. Amongother properties, compounds of this invention have been found to possessimproved binding affinity for hM₂ and hM₃ muscarinic receptor subtypes,have longer receptor half-lives, have a larger therapeutic window, orhave greater potency compared to related compounds. Accordingly,compounds of the invention are expected to be useful and advantageous astherapeutic agents for treating pulmonary disorders.

One aspect of the invention relates to compounds having formula I:

in salt or zwitterionic form, wherein:

a and b are independently 0 or an integer of from 1 to 5;

each R¹ and R² is independently selected from —C₁₋₄alkyl, —C₂₋₄alkenyl,—C₂₋₄alkynyl, —C₃₋₆cycloalkyl, cyano, halo, —OR^(a), —CH₂OH, —COOH,—C(O)O—C₁₋₄alkyl, —C(O)NR^(b)R^(c), —SR^(a), —S(O)R^(a), —S(O)₂R^(a),and —NR^(b)R^(c); where each R^(a) is independently selected fromhydrogen, —C₁₋₄alkyl, —C₂₋₄alkenyl, —C₂₋₄alkynyl and —C₃₋₆cycloalkyl;each R^(b) and R^(c) is independently selected from hydrogen,—C₁₋₄alkyl, —C₂₋₄alkenyl, —C₂₋₄alkynyl or —C₃₋₆cycloalkyl; or R^(b) andR^(c) together with the nitrogen atom to which they are attached form aC₃₋₆heterocycle; or two adjacent R¹ groups or two adjacent R² groups arejoined together to form —C₃₋₆alkylene, —C₂₋₄alkylene-O— or—O—C₁₋₄alkylene-O—;

R³ is selected from —C(O)NR^(3a)R^(3b), —C(O)O—C₁₋₄alkyl, —CN, —OH,—CH₂OH, and —CH₂NH₂;

R^(3a) and R^(3b) are independently selected from hydrogen, —C₁₋₆alkyl,—C₂₋₆alkenyl, —C₂₋₆alkynyl, —C₃₋₆cycloalkyl, —C₆₋₁₀aryl,—C₂₋₉heteroaryl, —C₃₋₆heterocycle, and —(CH₂)₁₋₂—R^(3c), where R^(3c) isselected from —OH, —O—C₁₋₆alkyl, —C₃₋₆cycloalkyl, —C₆₋₁₀aryl,—C₂₋₉heteroaryl, and —C₃₋₆heterocycle; or R^(3a) and R^(3b) togetherwith the nitrogen atom to which they are attached form a C₃₋₆heterocycleoptionally containing one additional heteroatom selected from nitrogen,oxygen or sulfur;

c is 0 or an integer of from 1 to 3;

each R⁴ is independently fluoro or —C₁₋₄alkyl;

d is 1 or 2, and

depicts an optional double bond;

R⁵ is selected from —C₁₋₆alkyl, —CH₂—C₂₋₆alkenyl, —CH₂—C₂₋₆alkynyl, and—CH₂COR^(5a); where R^(5a) is selected from —OH, —O—C₁₋₆alkyl, and—NR^(5b)R^(5c); and R^(5b) and R^(5c) are independently selected from Hand —C₁₋₆alkyl;

Q is —C₀₋₅alkylene-Q′-C₀₋₁alkylene-, wherein Q′ is selected from —CH₂—,—CH═CH—, —C≡C—, —O—, —S—, —S(O)—, —SO₂—, —SO₂—NR^(Q1)—, —NR^(Q1)—SO₂—,—C(O)—, —OC(O)—, —C(O)O—, —NR^(Q1)C(O)—, —C(O)NR_(Q1)—,—NR^(Q2)—C(O)—NR^(Q3)—, —NR^(Q2)—C(S)—NR^(Q3)—, —C═N—O—, —S—S—, and—C(═N—O—R^(Q4))—, where R^(Q1) is hydrogen or —C₁₋₁₄alkyl, R^(Q2) andR^(Q3) are independently selected from hydrogen, —C₁₋₄alkyl, and—C₃₋₆cycloalkyl, or R^(Q2) and R^(Q3) are taken together to form—C₂₋₄alkylene or —C₂₋₃alkenylene, and R^(Q4) is —C₁₋₄alkyl or benzyl;

e is 0 or an integer of from 1 to 5;

each R⁶ is independently selected from halo, —C₁₋₄alkyl,—C₀₋₄alkylene-OH, cyano, —C₀₋₂alkylene-COOH, —C(O)O—C₁₋₄alkyl,—O—C₁₋₄alkyl, —S—C₁₋₄alkyl, —NH—C(O)—C₁₋₄alkyl, —N(C₁₋₄alkyl)₂, and—N⁺(O)O;

wherein each alkyl, alkenyl, alkylene, alkynyl and cycloalkyl group inR¹⁻³, R^(3a-3c), R⁴⁻⁶, and R^(a)-R^(c) is optionally substituted with 1to 5 fluoro atoms; wherein each alkyl, alkenyl, and alkynyl group in R⁵is optionally substituted with 1 to 2 substituents independentlyselected from —O—C₁₋₆alkyl, —OH and phenyl; each cycloalkyl, aryl,heteroaryl and heterocycle group in R¹⁻², R^(3a-3c), and R^(a-c) isoptionally substituted with 1 to 3 substituents independently selectedfrom —C₁₋₄alkyl, —C₂₋₄alkenyl, —C₂₋₄alkynyl, cyano, halo, —O—C₁₋₄alkyl,—S—C₁₋₄alkyl, —S(O)(C₁₋₄alkyl), —S(O)₂(C₁₋₄alkyl), —NH₂, —NH(C₁₋₄alkyl)and —N(C₁₋₄alkyl)₂, wherein each alkyl, alkenyl and alkynyl group isoptionally substituted with 1 to 5 fluoro substituents; and each —CH₂—group in Q is optionally substituted with 1 or 2 substituentsindependently selected from —C₁₋₂alkyl, —OH and fluoro;

or a pharmaceutically acceptable salt thereof.

One aspect of the invention relates to quaternary ammonium compoundshaving formula I′:

or a pharmaceutically acceptable salt thereof, where X⁻ is an anion of apharmaceutically acceptable acid; and R¹⁻⁶, a-e, and Q are as definedabove. Another aspect of the invention relates to quaternary ammoniumcompounds having formula I′a:

or a pharmaceutically acceptable salt thereof, where R¹⁻², R^(3a-3b),R⁵⁻⁶, a, b, e, Q and X⁻ are as defined above. In one particularembodiment of formula I′a, Q is —C₂₋₅alkylene-Q′-. Still another aspectof the invention relates to quaternary ammonium compounds having formulaI′b:

or a pharmaceutically acceptable salt thereof, where R⁶, e, Q and X⁻ areas defined above. In one particular embodiment of formula I′b, Q is—C₂₋₅alkylene-Q′-. Another aspect of the invention relates to quaternaryammonium compounds having formula I:

or a pharmaceutically acceptable salt thereof, where R¹⁻², R^(3a-3b),R⁵⁻⁶, a, b, e, Q and X⁻ are as defined above. In one particularembodiment of formula I′c, Q is —C₂₋₅alkylene-Q′-.

Among the compounds of formula I, compounds of particular interest arethose having an inhibition dissociation constant (K_(i)) for binding tothe M₃ receptor subtype of less than or equal to 100 nM; in particularhaving a K_(i) less than or equal to 50 nM; more particularly having aK_(i) less than or equal to 10 nM; and even more particularly having aK_(i) less than or equal to 1.0 nM.

Another aspect of the invention relates to pharmaceutical compositionscomprising a pharmaceutically acceptable carrier and a compound of theinvention. Such compositions may optionally contain other therapeuticagents such as steroidal anti-inflammatory agents (e.g.,corticosteroids), β₂ adrenergic receptor agonists, phosphodiesterase-4inhibitors, and combinations thereof. Accordingly, in yet another aspectof the invention, a pharmaceutical composition comprises a compound ofthe invention, a second active agent, and a pharmaceutically acceptablecarrier. Another aspect of the invention pertains to a combination ofactive agents, comprising a compound of the invention and a secondactive agent. The compound of the invention can be formulated togetheror separately from the additional agent(s). When formulated separately,a pharmaceutically acceptable carrier may be included with theadditional agent(s). Thus, yet another aspect of the invention relatesto a combination of pharmaceutical compositions, the combinationcomprising: a first pharmaceutical composition comprising a compound ofthe invention and a first pharmaceutically acceptable carrier; and asecond pharmaceutical composition comprising a second active agent and asecond pharmaceutically acceptable carrier. This invention also relatesto a kit containing such pharmaceutical compositions, for example wherethe first and second pharmaceutical compositions are separatepharmaceutical compositions.

Compounds of the invention possess muscarinic receptor antagonistactivity, and are therefore expected to be useful as therapeutic agentsfor treating patients suffering from a disease or disorder that istreated by blocking the muscarinic receptor. Thus, one aspect of theinvention is directed to a method of producing bronchodilation in apatient, comprising administering to the patient abronchodilation-producing amount of a compound of the invention. Theinvention is also directed to method of treating a pulmonary disordersuch as chronic obstructive pulmonary disease or asthma, comprisingadministering to a patient a therapeutically effective amount of acompound of the invention. Another aspect of the invention relates to amethod for antagonizing a muscarinic receptor in a mammal comprisingadministering to the mammal, a muscarinic receptor-antagonizing amountof a compound of the invention.

Since compounds of the invention possess muscarinic receptor antagonistactivity, such compounds are also useful as research tools. Accordingly,one aspect of the invention pertains to a method of using a compound ofthe invention as a research tool, the method comprising conducting abiological assay using a compound of the invention. Compounds of theinvention can also be used to evaluate new chemical compounds. Thusanother aspect of the invention relates to a method of evaluating a testcompound in a biological assay, comprising: (a) conducting a biologicalassay with a test compound to provide a first assay value; (b)conducting the biological assay with a compound of the invention toprovide a second assay value; wherein step (a) is conducted eitherbefore, after or concurrently with step (b); and (c) comparing the firstassay value from step (a) with the second assay value from step (b).Exemplary biological assays include a muscarinic receptor binding assayand a bronchoprotection assay in a mammal. Still another aspect of theinvention is directed to a method of studying a biological system orsample comprising a muscarinic receptor, the method comprising: (a)contacting the biological system or sample with a compound of theinvention; and (b) determining the effects caused by the compound on thebiological system or sample.

The invention is also directed to processes and intermediates useful forpreparing compounds of the invention. Accordingly, another aspect of theinvention relates to a process of preparing compounds of the invention,comprising: (a) reacting a compound of formula II with a compound offormula III to produce a compound of formula IV, and reacting thecompound of formula IV with an organic substrate containing an R⁵ group;or (b) reacting a compound of formula V with a compound of formula III;or (c) reacting a compound of formula V with a compound of formula VI toproduce a compound of formula VII, and reacting the compound of formulaVII with a compound of formula VIII; and recovering the product in saltor zwitterionic form, to provide a compound of formula I or I′; whereincompounds of formula II-VIII are as defined herein. In other aspects,the invention is directed to products prepared by any of the processesdescribed herein.

Yet another aspect of the invention is directed to the use of a compoundof the invention for the manufacture of a medicament, especially for themanufacture of a medicament useful for treating a pulmonary disorder orfor antagonizing a muscarinic receptor in a mammal. Still another aspectof the invention pertains to the use of a compound of the invention as aresearch tool. Other aspects and embodiments of the invention aredisclosed herein.

DETAILED DESCRIPTION OF THE INVENTION

The invention is directed to compounds having formula I:

in salt or zwitterionic form, or a pharmaceutically acceptable saltthereof. More specifically, the invention is directed to quaternaryammonium compounds having formula I′:

or a pharmaceutically acceptable salt thereof, where X⁻ is an anion of apharmaceutically acceptable acid. The term “quaternary ammoniumcompound” refers to a compound that is derived from ammonium hydroxideor from an ammonium salt, wherein all four hydrogen atoms of the NH₄ ⁻ion have been replaced by organic groups.

As used herein, the term “compound of the invention” is intended toinclude compounds of formula I as well as the species embodied informulas I′, I′a, I′b, I′c, I′d, and I′e. The compounds of the inventionare quaternary ammonium salts and may be converted between differentsalt forms using state of the art methodologies, for example, using ionexchange chromatography. Also, the compounds can be obtained in the formof solvates, and such solvates are included within the scope of thisinvention. Accordingly, those skilled in the art will recognize thatreference to a compound herein, for example, reference to a “compound ofthe invention” includes reference to a compound of formula I as well asto any pharmaceutically acceptable salt forms and pharmaceuticallyacceptable solvates of that compound unless otherwise indicated.

The compounds of the invention may contain one or more chiral centersand so may exist in a number of stereoisomeric forms. When such chiralcenters are present, this invention is directed to racemic mixtures,pure stereoisomers (i.e., enantiomers or diastereomers),stereoisomer-enriched mixtures, and the like unless otherwise indicated.When a chemical structure is depicted without any stereochemistry, it isunderstood that all possible stereoisomers are encompassed by suchstructure. Thus, for example, the term “compound of formula I” isintended to include all possible stereoisomers of the compound.Similarly, when a particular stereoisomer is shown or named herein, itwill be understood by those skilled in the art that minor amounts ofother stereoisomers may be present in the compositions of this inventionunless otherwise indicated, provided that the utility of the compositionas a whole is not eliminated by the presence of such other isomers.Individual enantiomers may be obtained by numerous methods that are wellknown in the art, including chiral chromatography using a suitablechiral stationary phase or support, or by chemically converting theminto diastereomers, separating the diastereomers by conventional meanssuch as chromatography or recrystallization, then regenerating theoriginal enantiomers. Additionally, where applicable, all cis-trans orE/Z isomers (geometric isomers), tautomeric forms and topoisomeric formsof the compounds of this invention are included within the scope of thisinvention unless otherwise specified.

In particular, when d is 1, the compounds of formula I contain a chiralcenter at the carbon atom indicated by the symbol * in the followingpartial formula (shown without optional substituents for clarity):

In one embodiment of this invention, the carbon atom identified by thesymbol * has the (R) configuration. In this embodiment, compounds offormula I have the (R) configuration at the carbon atom identified bythe symbol * or are enriched in a stereoisomeric form having the (R)configuration at this carbon atom. In another embodiment, the carbonatom identified by the symbol * has the (S) configuration. In thisembodiment, compounds of formula I have the (S) configuration at thecarbon atom identified by the symbol * or are enriched in astereoisomeric form having the (S) configuration at this carbon atom.

The compounds of the invention, as well as those compounds used in theirsynthesis, may also include isotopically-labeled compounds, i.e., whereone or more atoms have been enriched with atoms having an atomic massdifferent from the atomic mass predominately found in nature. Examplesof isotopes that may be incorporated into the compounds of formula I,for example, include, but are not limited to, ²H, ³H, ¹³C, ¹⁴C, ¹⁵N, ¹⁸Oand ¹⁷O.

The compounds of the invention have been found to possess muscarinicreceptor antagonist activity. Among other properties, compounds of theinvention have been found to possess improved binding affinity for hM₂and hM₃ muscarinic receptor subtypes, have longer receptor half-lives,and have greater potency compared to related compounds, and are expectedto be useful as therapeutic agents for treating pulmonary disorders.

The nomenclature used herein to name the compounds of the invention isillustrated in the Examples herein. This nomenclature has been derivedusing the commercially-available AutoNom software (MDL, San Leandro,Calif.).

Representative Embodiments

The following substituents and values are intended to providerepresentative examples of various aspects and embodiments of theinvention. These representative values are intended to further defineand illustrate such aspects and embodiments and are not intended toexclude other embodiments or to limit the scope of the invention. Inthis regard, the representation that a particular value or substituentis preferred is not intended in any way to exclude other values orsubstituents from the invention unless specifically indicated.

The values for a and b are independently 0, 1, 2, 3, 4 or 5;particularly independently 0, 1 or 2; and even more particularly 0 or 1.In one embodiment, a is 0 or 1. In another embodiment, b is 0. In oneembodiment, both a and b are 0. In another embodiment, a is 1 and b is0.

When present, each R¹ and R² may be at the 2, 3, 4, 5 or 6-position ofthe phenyl ring to which it is attached. Each R¹ and R² is independentlyselected from —C₁₋₄alkyl, —C₂₋₄alkenyl, —C₂₋₄alkynyl, —C₃₋₆cycloalkyl,cyano, halo, —OR^(a), —CH₂OH, —COOH, —C(O)—O—C₁₋₄alkyl,—C(O)NR^(b)R^(c), —SR^(a), —S(O)R^(a), —S(O)₂R^(a), and —NR^(b)R^(c).Each R^(a) is independently selected from hydrogen, —C₁₋₄alkyl,—C₂₋₄alkenyl, —C₂₋₄alkynyl and —C₃₋₆cycloalkyl. Each R^(b) and R^(c) isindependently selected from hydrogen, —C₁₋₄alkyl, —C₂₋₄alkenyl,—C₂₋₄alkynyl or —C₃₋₆cycloalkyl. Alternatively, R^(b) and R^(c) togetherwith the nitrogen atom to which they are attached form aC₃₋₆heterocycle. In another embodiment, two adjacent R¹ groups or twoadjacent R² groups are joined together to form —C₃₋₆alkylene,—C₂₋₄alkylene-O— or —O—C₁₋₄alkylene-O—. In one embodiment, R¹ or R² areindependently selected from —C₁₋₄alkyl (e.g. methyl, ethyl, n-propyl,isopropyl), fluoro, chloro, —OR^(a) (e.g., hydroxy, methoxy, ethoxy),—CH₂OH, —COOH, —C(O)—O—C₁₋₄alkyl (e.g., —COOCH₃), and —NR^(b)R^(c)(e.g., NH₂). In another specific embodiment, R¹ is selected from—C₁₋₄alkyl and —OR^(a).

Each of the aforementioned alkyl, alkenyl, alkylene, alkynyl andcycloalkyl groups in R¹, R², R^(a), R^(b) and R^(c) may be substitutedwith 1 to 5 fluoro atoms. For example, R¹ or R² can be —C₁₋₄alkyl suchas difluoromethyl, trifluoromethyl, and 2,2,2-trifluoroethyl, or—OR^(a), where R^(a) is difluoromethyl or trifluoromethyl. In addition,each cycloalkyl group in R¹, R², R^(a), R^(b) and R^(c) may besubstituted with 1 to 3 substituents independently selected from—C₁₋₄alkyl, —C₂₋₄alkenyl, —C₂₋₄alkynyl, cyano, halo, —O—C₁₋₄alkyl,—S—C₁₋₄alkyl, —S(O)(C₁₋₄alkyl), —S(O)₂(C₁₋₄alkyl), —NH₂, —NH(C₁₋₄alkyl)and —N(C₁₋₄alkyl)₂, where each of these alkyl, alkenyl and alkynylgroups can be substituted with 1 to 5 fluoro substituents.

R³ is selected from —C(O)NR^(3a)R^(3b), —C(O)O—C₁₋₄alkyl, —CN, —OH,—CH₂OH, and —CH₂NH₂. R^(3a) and R^(3b) are independently selected fromhydrogen, —C₁₋₆alkyl, —C₂₋₆alkenyl, —C₂₋₆alkynyl, —C₃₋₆cycloalkyl,—C₆₋₁₀aryl, —C₂₋₉heteroaryl, —C₃₋₆heterocycle, and —(CH₂)₁₋₂—R^(3c).R^(3c) is selected from —OH, —O—C₁₋₆alkyl, —C₃₋₆cycloalkyl, —C₆₋₁₀aryl,—C₂₋₉heteroaryl, and —C₃₋₆heterocycle. In one embodiment, R³ is—C(O)NR^(3a)R^(3b). In one embodiment, R^(3a) and R^(3b) areindependently hydrogen or —C₁₋₄alkyl. In another embodiment R^(3a) andR^(3b) are independently hydrogen or —C₁₋₂alkyl, such as methyl andethyl. In yet another embodiment, R^(3a) and R^(3b) are both hydrogen.Alternatively, R^(3a) and R^(3b) together with the nitrogen atom towhich they are attached form a C₃₋₆heterocycle optionally containing oneadditional heteroatom selected from nitrogen, oxygen or sulfur.Representative heterocyclic rings include, but are not limited to,pyrrolidin-1-yl, piperidin-1-yl, piperazin-1-yl,4-C₁₋₄alkylpiperazin-1-yl, morpholin-4-yl and thiomorpholin-4-yl.

Each of the aforementioned alkyl, alkenyl, alkynyl and cycloalkyl groupsin R³ and R^(3a-3c) may be substituted with 1 to 5 fluoro atoms. Forexample, R³ can be —CF₃. In addition, each aryl, cycloalkyl, heteroaryland heterocycle group in R^(3a-3c) may be substituted with 1 to 3substituents independently selected from —C₁₋₄alkyl, —C₂₋₄alkenyl,—C₂₋₄alkynyl, cyano, halo, —O—C₁₋₄alkyl, —S—C₁₋₄alkyl, —S(O)(C₁₋₄alkyl),—S(O)₂(C₁₋₄alkyl), —NH₂, —NH(C₁₋₄alkyl) and —N(C₁₋₄alkyl)₂, where eachof these alkyl, alkenyl and alkynyl groups can be substituted with 1 to5 fluoro substituents.

The value for c is 0, 1, 2, or 3; particularly 0, 1 or 2; and even moreparticularly 0 or 1. In one embodiment, c is 0. In another embodiment,each of a, b and c represents 0.

When present, each R⁴ is independently selected fluoro or —C₁₋₄alkyl.When more than one R⁴ substituent is present, i.e., c is 2 or 3, thesubstituents can be on the same or on different carbon atoms. ExemplaryR⁴ groups include, but are not limited to, methyl, ethyl, and fluoro.The alkyl group in R⁴ may be substituted with 1 to 5 fluoro atoms. Forexample, R⁴ can be difluoromethyl or trifluoromethyl.

The value for d is 1 or 2, and depicts an optional double bond. In oneembodiment, d is 1 and the double bond is absent, i.e., when the ringdefined by “d” is a pyrrolidine ring. In one embodiment, thestereocenter at the 3-position of the pyrrolidine ring (i.e., the carbonatom bearing the 1-carbamoyl-1,1-diphenylmethyl group) has the (S)stereochemistry. In another embodiment, this stereocenter has the (R)stereochemistry. In one embodiment, d is 1 and the double bond ispresent, i.e., when the ring defined by “d” is a tetrahydropyridinering.

R⁵ is selected from —C₁₋₆alkyl, —CH₂—C₂₋₆alkenyl, —CH₂—C₂₋₆alkynyl, and—CH₂COR^(a). In some embodiments, R⁵ may be absent. R^(5a) is selectedfrom —OH, —O—C₁₋₆alkyl, and —NR^(5b)R^(5c), where R^(5b) and R^(5c) areindependently selected from H and —C₁₋₆alkyl. In one embodiment, R⁵ is—C₁₋₆alkyl such as —CH₃ or —CH₂CH₃; and in a particular embodiment R⁵ is—CH₃. Each of the aforementioned alkyl, alkenyl, and alkynyl groups inR⁵ may be substituted with 1 to 5 fluoro atoms. For example, R⁵ can be—CF₃. Each of the aforementioned alkyl, alkenyl, and alkynyl groups inR⁵ may also be substituted with 1 to 2-O—C₁₋₆alkyl, —OH and phenylgroups.

Q is —C₀₋₅alkylene-Q′-C₀₋₁alkylene-. Q′ is selected from —CH₂—, —CH═CH—,—C≡C—, —O—, —S—, —S(O)—, —SO₂—, —SO₂—NR^(Q1)—, —NR^(Q1)—SO₂—, —C(O)—,—OC(O)—, —C(O)O—, —NR^(Q1)C(O)—, —C(O)NR^(Q1)—, —NR^(Q2)—C(O)—NR^(Q3)—,—NR^(Q2)—C(S)—NR^(Q3)—, —C═N—O—, —S—S—, and —C(═N—O—R^(Q4))—. R^(Q1) ishydrogen or —C₁₋₄alkyl. In one particular embodiment, R^(Q1) ishydrogen. R^(Q2) and R^(Q3) are independently selected from hydrogen,—C₁₋₄alkyl, and —C₃₋₆cycloalkyl, or they can be taken together to form—C₂₋₄alkylene or —C₂₋₃alkenylene. In one particular embodiment, R^(Q2)and R^(Q3) are both hydrogen. R^(Q4) is —C₁₋₄alkyl or benzyl. In oneembodiment, R^(Q4) is —C₁₋₄alkyl such as —CH₃. In one particularembodiment, R^(Q4) is benzyl. In one particular embodiment, Q′ isselected from —CH₂—, —O—, —S—, —S(O)—, —C(O)—, —OC(O)—, —C(O)O—,—NR^(Q1)C(O)—, —C(O)NR^(Q1)—, —NR^(Q2)—C(O)—NR^(Q3)—,—NR^(Q2)—C(S)—NR^(Q3)—, and —C(═N—O—R^(Q4))—. In another particularembodiment, Q′ is selected from —O—, —S—, —C(O)—, and —OC(O)—.

The linker connecting Q′ to the N⁺ atom, —C₀₋₅alkylene-, may be a bond(0 carbon atoms) or have 1, 2, 3, 4, or 5 carbon atoms. In oneparticular embodiment, this linker is —C₂₋₅alkylene-; in anotherparticular embodiment, —C₁₋₃alkylene-; and in yet another embodiment—C₃alkylene-. Each —CH₂— group in the —C₀₋₅alkylene- linker may besubstituted with 1 or 2 substituents independently selected from—C₁₋₂alkyl, —OH and fluoro. In one particular embodiment, the linker is—(CH₂)₃— and one —CH₂— group is substituted with —OH, for example—CH₂—CH(OH)—CH₂—.

The linker connecting Q′ to the phenyl ring, —C₀₋₁alkylene-, may be abond (0 carbon atoms) or have 1 carbon atom. In one particularembodiment, this linker is a bond. Each —CH₂— group in the—C₀₋₁alkylene- linker may be substituted with 1 or 2 substituentsindependently selected from —C₁₋₂alkyl, —OH and fluoro.

In specific embodiments, —C₀₋₅alkylene-Q′-C₀₋₁alkylene- is one of thefollowing: —(CH₂)₂—, —(CH₂)₃—, —(CH₂)₄—, —(CH₂)₅—, —(CH₂)₆—,—(CH₂)₂—CH═CH—, —(CH₂)₂—C≡C—, —(CH₂)₂—O—CH₂—, —(CH₂)₃—O—,—CH₂—CH(OH)—CH₂—O—, —(CH₂)₃—O—CH₂—, —(CH₂)₃—S—, —(CH₂)₃—S(O)—,—(CH₂)₃—SO₂—, —(CH₂)₃—C(O)—, —(CH₂)₂—OC(O)—, —(CH₂)₂—C(O)O—,—CH₂—C(O)O—CH₂—, —(CH₂)₂—NR^(Q1)C(O)—, —CH₂—C(O)NR^(Q1)—CH₂,—(CH₂)₂—NR^(Q2)—C(O)—NR^(Q3)—, —CH₂—C═N—O—, —(CH₂)₂—S—S—, and—(CH₂)₃—C(═N—O—R^(Q4))—, where R^(Q1), R^(Q2) and R^(Q3) are hydrogen,and R^(Q4) is —C₁₋₄alkyl or benzyl.

The value for e is 0, 1, 2, 3, 4 or 5. In one embodiment, e is 0, 1 or2.

When present, each R⁶ is independently selected from halo, —C₁₋₄alkyl,—C₀₋₄-alkylene-OH, cyano, —C₀₋₂alkylene-COOH, —C(O)O—C₁₋₄alkyl,—O—C₁₋₄alkyl, —S—C₁₋₄alkyl, —NH—C(O)—C₁₋₄alkyl, —N(C₁₋₄alkyl)₂, and—N⁺(O)O. In one particular embodiment, R⁶ is selected from halo,—C₁₋₄alkyl, —OH, cyano, —C(O)O—C₁₋₄alkyl, —O—C₁₋₄alkyl, —S—C₁₋₄alkyl,—NH—C(O)—C₁₋₄alkyl, —N(CH₃)₂, and —N⁺(O)O. In one embodiment, R⁶ is halosuch as fluoro, chloro and bromo. In a particular embodiment e is 1 andR⁶ is fluoro or chloro; in another embodiment, e is 2 and both R⁶ groupsare fluoro, or both R⁶ groups are chloro, or one R⁶ group is fluoro andthe other R⁶ group is chloro. In another embodiment, R⁶ is —C₁₋₄alkylsuch as —CH₃. In one embodiment, R⁶ is —OH. In one embodiment, R⁶ is—C₁₋₄alkylene-OH. In one embodiment, R⁶ is cyano. In one embodiment, R⁶is —C₀₋₂alkylene-COOH such as —COOH. In one embodiment, R⁶ is—C(O)O—C₁₋₄alkyl such as —C(O)O—CH₃. In one embodiment, R⁶ is—O—C₁₋₄alkyl such as —O—CH₃. In one embodiment, R⁶ is —S—C₁₋₄alkyl suchas —S—CH₃. In one embodiment, R⁶ is —NH—C(O)—C₁₋₄alkyl such as—NH—C(O)—CH₃. In yet another embodiment, R⁶ is —N(C₁₋₄alkyl)₂ such as—N(CH₃)₂. In one embodiment, R⁶ is —N⁺(O)O. The alkyl group in R⁶ may besubstituted with 1 to 5 fluoro atoms. For example, R⁶ can be —CF₃ or—OCF₃.

X⁻ is an anion of a pharmaceutically acceptable acid. The term “anion ofa pharmaceutically acceptable acid” is used to refer to an anioniccounterion of a pharmaceutically acceptable acid. Examples ofpharmaceutically acceptable inorganic acids include, by way ofillustration and not limitation, boric, carbonic, hydrohalic(hydrobromic, hydrochloric, hydrofluoric or hydroiodic), nitric,phosphoric, sulfamic and sulfuric acids, and hydroxide. Examples ofpharmaceutically acceptable organic acids include, by way ofillustration and not limitation, aliphatic hydroxyl acids (e.g., citric,gluconic, glycolic, lactic, lactobionic, malic, and tartaric acids),aliphatic monocarboxylic acids (e.g., acetic, butyric, formic, propionicand trifluoroacetic acids), aromatic carboxylic acids (e.g., benzoic,p-chlorobenzoic, diphenylacetic, gentisic, hippuric, and triphenylaceticacids), amino acids (e.g., aspartic and glutamic acids), aromatichydroxyl acids (e.g., o-hydroxybenzoic, p-hydroxybenzoic,1-hydroxynaphthalene-2-carboxylic and 3-hydroxynaphthalene-2-carboxylicacids), ascorbic, dicarboxylic acids (e.g., fumaric, maleic, oxalic andsuccinic acids), glucoronic, mandelic, mucic, nicotinic, orotic, pamoic,pantothenic, sulfonic acids (e.g., benzenesulfonic, camphosulfonic,edisylic, ethanesulfonic, isethionic, methanesulfonic,naphthalenesulfonic, naphthalene-1,5-disulfonic,naphthalene-2,6-disulfonic and p-toluenesulfonic acids), xinafoic acid,and the like. In one embodiment, the pharmaceutically acceptable acid isselected from acetic, benzenesulfonic, benzoic, butyric,p-chlorobenzoic, citric, diphenylacetic, formic, hydrobromic,hydrochloric, hydrofluoric, hydroiodic, o-hydroxybenzoic,p-hydroxybenzoic, 1-hydroxynaphthalene-2-carboxylic,3-hydroxynaphthalene-2-carboxylic, lactic, malic, maleic,methanesulfonic, nitric, phosphoric, propionic, succinic, sulfuric,tartaric, trifluoroacetic, and triphenylacetic acids. In anotherembodiment the pharmaceutically acceptable acid is selected fromhydrobromic, hydroiodic, and trifluoroacetic acids. In one embodiment,the anion is selected from acetate, benzenesulfonate, benzoate, bromide,butyrate, chloride, p-chlorobenzoate, citrate, diphenylacetate, formate,fluoride, o-hydroxybenzoate, p-hydroxybenzoate,1-hydroxynaphthalene-2-carboxylate, 3-hydroxynaphthalene-2-carboxylate,iodide, lactate, malate, maleate, methanesulfonate, nitrate, phosphate,propionate, succinate, sulfate, tartrate, trifluoroacetate, bi- andtriphenylacetate. In yet another embodiment, the anion is selected frombromide, iodide and trifluoroacetate.

In one embodiment, R³ is —C(O)NR^(3a)R^(3b), c is 0 and d is 1. Thus,the invention is also directed to quaternary ammonium compounds havingformula I′a:

or a pharmaceutically acceptable salt thereof; where R¹⁻², R^(3a-3b),R⁵⁻⁶, a, b, e, Q and X⁻ are as defined for formula I. In anotherembodiment, R³ is —C(O)NH₂, R⁵ is —CH₃, a, b and c are 0 and d is 1.Thus, the invention is also directed to quaternary ammonium compoundshaving formula I′b:

or a pharmaceutically acceptable salt thereof; where R⁶, e, Q and X⁻ areas defined for formula I. In another embodiment, R³ is —C(O)NH₂, R⁵ is—CH₃, a, b and c are 0 and d is 2. Thus, the invention is also directedto quaternary ammonium compounds having formula I′c:

or a pharmaceutically acceptable salt thereof; where R¹⁻², R^(3a-3b),R⁵⁻⁶, a, b, e, Q and X⁻ are as defined for formula I.

The invention is also directed to quaternary ammonium compounds havingformula I′d:

or a pharmaceutically acceptable salt thereof; where: a and b areindependently 0 or 1; each R¹ and R² is —OR^(a), where R^(a) ishydrogen; Q is selected from: —(CH₂)₂—, —(CH₂)₃—, —(CH₂)₄—, —(CH₂)₅—,—(CH₂)₆—, —(CH₂)₂—CH═CH—, —(CH₂)₂—C≡C—, —(CH₂)₂—O—CH₂—, —(CH₂)₃—O—,—CH₂—CH(OH)—CH₂—O—, —(CH₂)₃—O—CH₂—, —(CH₂)₃—S—, —(CH₂)₃—S(O)—,—(CH₂)₃—SO₂—, —(CH₂)₃—C(O)—, —(CH₂)₂—OC(O)—, —(CH₂)₂—C(O)O—,—CH₂—C(O)O—CH₂—, —(CH₂)₂—NR^(Q1)C(O)—, —CH₂—C(O)NR^(Q1)—CH₂,—(CH₂)₂—NR^(Q2)—C(O)—NR^(Q3)—, —CH₂—C═N—O—, —(CH₂)₂—S—S—, and—(CH₂)₃—C(═N—O—R^(Q4))—, where R^(Q1), R^(Q2) and R^(Q3) are hydrogen,and R^(Q4) is —C₁₋₄alkyl or benzyl; e is 0, 1 or 2; each R⁶ isindependently selected from halo, —C₁₋₄alkyl, —C₀₋₄alkylene-OH, cyano,—C(O)O—C₁₋₄alkyl, —O—C₁₋₄alkyl, —S—C₁₋₄alkyl, —NH—C(O)—C₁₋₄alkyl,—N(C₁₋₄alkyl)₂, and —N⁺(O)O. The moieties may be optionally substitutedas described for formula I, and in one particular embodiment, the alkylin R⁶ is optionally substituted with 3 fluoro atoms, and one —CH₂— groupin Q is optionally substituted with —OH.

In another embodiment, the invention is directed to quaternary ammoniumcompounds having formula I′e:

or a pharmaceutically acceptable salt thereof; where a is 0 or 1; R¹ is—OR^(a), where R^(a) is hydrogen; Q is selected from: —(CH₂)₄—,—(CH₂)₂—CH═CH—, —(CH₂)₂—C≡C—, —(CH₂)₃—O—, —(CH₂)₃—S—, —(CH₂)₃—C(O)—, and—(CH₂)₂—OC(O)—; e is 0, 1 or 2; each R⁶ is independently selected fromhalo, —C₁₋₄alkyl, —OH, and —S—C₁₋₄alkyl. While all of the moieties maybe optionally substituted as described for formula I, in one particularembodiment, the compound is not optionally substituted.

In still another embodiment, the invention is directed to quaternaryammonium compounds having formula I″a, I″b, I″c, or I″d:

or a pharmaceutically acceptable salt thereof, where X⁻ is an anion of apharmaceutically acceptable acid. In one particular embodiment, theinvention is directed to compounds of formula I″a.

A particular group of compounds of formula I are those disclosed in U.S.Provisional Application No. 60/903,110, filed on Feb. 23, 2007. Thisgroup includes compounds of formula I′″:

in salt or zwitterionic form, wherein: a′ and b′ are independently 0 oran integer of from 1 to 5; each R¹′ and R²′ is independently selectedfrom —C₁₋₄alkyl, —C₂₋₄alkenyl, —C₂₋₄alkynyl, —C₃₋₆cycloalkyl, cyano,halo, —OR^(a)′, —SR^(a)′, —S(O)R^(a)′, —S(O)₂R^(a)′ and —NR^(b)′R^(c)′;where each R^(a)′ is independently selected from hydrogen, —C₁₋₄alkyl,—C₂₋₄alkenyl, —C₂₋₄alkynyl and —C₃₋₆cycloalkyl; each R^(b)′ and R^(c)′is independently selected from hydrogen, —C₁₋₄alkyl, —C₂₋₄alkenyl,—C₂₋₄alkynyl or —C₃₋₆cycloalkyl; or R^(b)′ and R^(c)′ together with thenitrogen atom to which they are attached form a C₃₋₆heterocycle; or twoadjacent R¹′ groups or two adjacent R²′ groups are joined together toform —C₃₋₆alkylene, —C₂₋₄alkylene-O— or —O—C₁₋₄alkylene-O—; R³′ isselected from —C(O)NR^(3a)′R^(3b)′, —C(O)O—C₁₋₄alkyl, —CN, —OH, —CH₂OH,and —CH₂NH₂; where R^(3a)′ and R^(3b)′ are independently selected fromhydrogen, —C₁₋₆alkyl, —C₂₋₆alkenyl, —C₂₋₆alkynyl, —C₃₋₆cycloalkyl,—C₆₋₁₀aryl, —C₂₋₉heteroaryl, —C₃₋₆heterocycle, and —(CH₂)₁₋₂—R^(3c)′,where R^(3c′) is selected from —OH, —O—C₁₋₆alkyl, —C₃₋₆cycloalkyl,—C₆₋₁₀aryl, —C₂₋₉heteroaryl, and —C₃₋₆heterocycle; or R^(3a)′ andR^(3b)′ together with the nitrogen atom to which they are attached forma C₃₋₆heterocycle optionally containing one additional heteroatomselected from nitrogen, oxygen or sulfur; c′ is 0 or an integer of from1 to 3; each R⁴′ is independently fluoro or —C₁₋₄alkyl; d′ is 1 or 2,and

depicts an optional double bond; R⁵′ is selected from —C₁₋₆alkyl,—CH₂—C₂₋₆alkenyl, CH₂—C₂₋₆alkynyl, and —CH₂COR^(5a′); where R^(5a′) isselected from —OH, —O—C₁₋₆alkyl, and —NR^(5b′)R^(5c′); and R^(5b′) andR^(5c)′ are independently selected from H and —C₁₋₁₆alkyl; Q′ isselected from —CH₂—, —O—, —S—, —S(O)—, —SO₂—, —SO₂—NR^(Q1)′—,—NR^(Q1)′—SO₂—, —C(O)—, —OC(O)—, —C(O)O—, —NR^(Q1)′C(O)—,—C(O)NR^(Q1)′—, NR^(Q2)′—C(O)—NR^(Q3)′—, —NR^(Q2)′—C(S)—NR^(Q3)′—,—CH(OH)—, and —C(═N—O—R^(Q4)′)—, where R^(Q1)′ is hydrogen or—C₁₋₄alkyl, R^(Q2)′ and R^(Q3)′ are independently selected fromhydrogen, —C₁₋₄alkyl, and —C₃₋₆cycloalkyl, or R^(Q2)′ and R^(Q3)′ aretaken together to form —C₂₋₄alkylene or —C₂₋₃alkenylene, and R^(Q4)′ is—C₁₋₄alkyl or benzyl; e′ is 0 or an integer of from 1 to 5; each R⁶′ isindependently selected from halo, —C₁₋₄alkyl, —OH, —C₁₋₄alkyl-OH, cyano,—COOH, —C(O)O—C₁₋₄alkyl, —C₁₋₄alkoxy, —S—C₁₋₄alkyl, —NH—C(O)—C₁₋₄alkyl,and —N⁺(O)O; wherein each alkyl, alkenyl, alkylene, alkynyl andcycloalkyl group in R¹′⁻³′, R^(3a)′^(-3c)′, R⁴′⁻⁶′, and R^(a-c)′ isoptionally substituted with 1 to 5 fluoro atoms; wherein each alkyl,alkenyl, and alkynyl group in R⁵′ is optionally substituted with 1 to 2substituents independently selected from —C₁₋₄alkoxy, —OH and phenyl;each cycloalkyl, aryl, heteroaryl and heterocycle group in R¹′²′,R^(3a)′⁻³′, and R^(a)′^(-c)′ is optionally substituted with 1 to 3substituents independently selected from —C₁₋₄alkyl, —C₂₋₄alkenyl,—C₂₋₄alkynyl, cyano, halo, —O—C₁₋₄alkyl, —S—C₁₋₄alkyl, —S(O)(C₁₋₄alkyl),—S(O)₂(C₁₋₄alkyl), —NH₂, —NH(C₁₋₄alkyl) and —N(C₁₋₄alkyl)₂, wherein eachalkyl, alkenyl and alkynyl group is optionally substituted with 1 to 5fluoro substituents; and each —CH₂— group in —(CH₂)₀₋₅— is optionallysubstituted with 1 or 2 substituents independently selected from—C₁₋₁₂alkyl, —OH and fluoro; or a pharmaceutically acceptable saltthereof.

In addition, particular compounds of formula I that are of interestinclude those set forth in the Examples below, as well as thepharmaceutically acceptable salts thereof.

DEFINITIONS

When describing the compounds, compositions, methods and processes ofthe invention, the following terms have the following meanings unlessotherwise indicated. Additionally, as used herein, the singular forms“a,” “an” and “the” include the corresponding plural forms unless thecontext of use clearly dictates otherwise. The terms “comprising”,“including,” and “having” are intended to be inclusive and mean thatthere may be additional elements other than the listed elements.

The term “alkyl” means a monovalent saturated hydrocarbon group whichmay be linear or branched. Unless otherwise defined, such alkyl groupstypically contain from 1 to 10 carbon atoms and include, for example—C₁₋₂alkyl, —C₁₋₄alkyl, and —C₁₋₆alkyl. Representative alkyl groupsinclude, by way of example, methyl, ethyl, n-propyl, isopropyl, n-butyl,sec-butyl, isobutyl, tert-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl,n-nonyl, n-decyl and the like.

When a specific number of carbon atoms is intended for a particular termused herein, the number of carbon atoms is shown preceding the term assubscript. For example, the term “—C₁₋₆alkyl” means an alkyl grouphaving from 1 to 6 carbon atoms, where the carbon atoms are in anyacceptable configuration.

The term “alkylene” means a divalent saturated hydrocarbon group thatmay be linear or branched. Unless otherwise defined, such alkylenegroups typically contain from 1 to 10 carbon atoms and include, forexample, C₀₋₁alkylene, C₀₋₅alkylene, C₁₋₄alkylene, C₂₋₄alkylene,C₂₋₅alkylene, and C₃₋₆alkylene. Representative alkylene groups include,by way of example, methylene, ethane-1,2-diyl (“ethylene”),propane-1,2-diyl, propane-1,3-diyl, butane-1,4-diyl, pentane-1,5-diyland the like. It is understood that when the alkylene term include zerocarbons such as C₀₋₁alkylene or C₀₋₅alkylene, such terms are intended toinclude a single bond.

The term “alkenyl” means a monovalent unsaturated hydrocarbon groupwhich may be linear or branched and which has at least one, andtypically 1, 2 or 3, carbon-carbon double bonds. Unless otherwisedefined, such alkenyl groups typically contain from 2 to carbon atomsand include, for example, —C₂₋₄alkenyl and —C₂₋₆alkenyl. Representativealkenyl groups include, by way of example, ethenyl, n-propenyl,isopropenyl, n-but-2-enyl, n-hex-3-enyl and the like. The term“alkenylene” means a divalent alkenyl group, and exemplary alkenylenegroups include —C₂₋₃alkenylene.

The term “alkoxy” means a monovalent group of the formula —O-alkyl,where alkyl is as defined herein. Unless otherwise defined, suchalkylene groups typically contain from 1 to 10 carbon atoms and include,for example, —O—C₁₋₄alkyl and —O—C₁₋₆alkyl. Representative alkoxy groupsinclude, by way of example, methoxy, ethoxy, n-propoxy, isopropoxy,n-butoxy, sec-butoxy, isobutoxy, tert-butoxy and the like.

The term “alkynyl” means a monovalent unsaturated hydrocarbon groupwhich may be linear or branched and which has at least one, andtypically 1, 2 or 3, carbon-carbon triple bonds. Unless otherwisedefined, such alkynyl groups typically contain from 2 to 10 carbon atomsand include, for example, —C₂₋₄alkynyl and —C₂₋₆alkynyl. Representativealkynyl groups include, by way of example, ethynyl, n-propynyl,n-but-2-ynyl, n-hex-3-ynyl and the like. The term “alkynylene” means adivalent alkynyl group, and exemplary alkynylene groups include—C₂₋₃alkenylene.

The term “amino-protecting group” means a protecting group suitable forpreventing undesired reactions at an amino group. Representativeamino-protecting groups include, but are not limited to,tert-butoxycarbonyl (BOC), trityl (Tr), benzyloxycarbonyl (Cbz),9-fluorenylmethoxycarbonyl (Fmoc), formyl, trimethylsilyl (TMS),tert-butyldimethylsilyl (TBS), and the like.

The term “aryl” means a monovalent aromatic hydrocarbon having a singlering (i.e., phenyl) or fused rings (i.e., naphthalene). Unless otherwisedefined, such aryl groups typically contain from 6 to 10 carbon ringatoms and include, for example, —C₆₋₁₀aryl. Representative aryl groupsinclude, by way of example, phenyl and naphthalene-1-yl,naphthalene-2-yl, and the like.

The term “cycloalkyl” means a monovalent saturated carbocyclichydrocarbon group. Unless otherwise defined, such cycloalkyl groupstypically contain from 3 to 10 carbon atoms and include, for example,—C₃₋₆cycloalkyl. Representative cycloalkyl groups include, by way ofexample, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and the like.

The term “divalent hydrocarbon group” means a divalent hydrocarbon groupwhich is composed primarily of carbon and hydrogen atoms and whichoptionally contains one or more heteroatoms. Such divalent hydrocarbongroups may be branched or unbranched, saturated or unsaturated, acyclicor cyclic, aliphatic or aromatic, or combinations thereof. The divalenthydrocarbon group can optionally contain heteroatoms incorporated intothe hydrocarbon chain or as substituents attached to the hydrocarbonchain.

The term “halo” means fluoro, chloro, bromo and iodo.

The term “heteroaryl” means a monovalent aromatic group having a singlering or two fused rings and containing in the ring at least oneheteroatom (typically 1 to 3 heteroatoms) selected from nitrogen, oxygenor sulfur. Unless otherwise defined, such heteroaryl groups typicallycontain from 5 to 10 total ring atoms and include, for example,—C₂₋₉heteroaryl. Representative heteroaryl groups include, by way ofexample, monovalent species of pyrrole, imidazole, thiazole, oxazole,furan, thiophene, triazole, pyrazole, isoxazole, isothiazole, pyridine,pyrazine, pyridazine, pyrimidine, triazine, indole, benzofuran,benzothiophene, benzoimidazole, benzthiazole, quinoline, isoquinoline,quinazoline, quinoxaline and the like, where the point of attachment isat any available carbon or nitrogen ring atom.

The term “heterocycle” or “heterocyclic” means a monovalent saturated orunsaturated (non-aromatic) group having a single ring or multiplecondensed rings and containing in the ring at least one heteroatom(typically 1 to 3 heteroatoms) selected from nitrogen, oxygen or sulfur.Unless otherwise defined, such heterocyclic groups typically containfrom 2 to 9 total ring carbon atoms and include, for example,—C₃₋₆heterocycle. Representative heterocyclic groups include, by way ofexample, monovalent species of pyrrolidine, imidazolidine, pyrazolidine,piperidine, 1,4-dioxane, morpholine, thiomorpholine, piperazine,3-pyrroline and the like, where the point of attachment is at anyavailable carbon or nitrogen ring atom.

The term “leaving group” means a functional group or an atom that can bedisplaced by another functional group or atom in a substitutionreaction, such as a nucleophilic substitution reaction. By way ofexample, representative leaving groups include, but are not limited to,chloro, bromo and iodo groups; sulfonic ester groups, such as mesylate,tosylate, brosylate, nosylate and the like; and acyloxy groups, such asacetoxy, trifluoroacetoxy and the like.

The term “optionally substituted” means that group in question may beunsubstituted or it may be substituted one or several times, such as 1to 3 times or 1 to 5 times. For example, an alkyl group that is“optionally substituted” with 1 to 5 fluoro atoms, may be unsubstituted,or it may contain 1, 2, 3, 4, or 5 fluoro atoms.

The term “pharmaceutically acceptable” refers to a material that is notbiologically or otherwise undesirable. For example, the term“pharmaceutically acceptable carrier” refers to a material that can beincorporated into a composition and administered to a patient withoutcausing undesirable biological effects or interacting in a deleteriousmanner with other components of the composition. Such pharmaceuticallyacceptable materials typically have met the required standards oftoxicological and manufacturing testing, and include those materialsidentified as suitable inactive ingredients by the U.S. Food and Drugadministration.

The term “solvate” means a complex or aggregate formed by one or moremolecules of a solute, e.g., a compound of formula I or apharmaceutically acceptable salt thereof, and one or more molecules of asolvent. Such solvates are typically crystalline solids having asubstantially fixed molar ratio of solute and solvent. Representativesolvents include, by way of example, water, methanol, ethanol,isopropanol, acetic acid and the like. When the solvent is water, thesolvate formed is a hydrate.

The term “therapeutically effective amount” means an amount sufficientto effect treatment when administered to a patient in need of treatment.In particular, an “effective” amount is that amount needed to obtain thedesired result, and a “therapeutically effective” amount is that amountneeded to obtain the desired therapeutic effect. For example, forantagonizing a muscarinic receptor, an “effective amount” is amuscarinic-receptor-antagonizing amount. Similarly, a therapeuticallyeffective amount for treating chronic obstructive pulmonary disease(COPD) is that amount that will achieve the desired therapeutic result,which may be disease prevention, amelioration, suppression oralleviation.

The term “treating” or “treatment” as used herein means the treating ortreatment of a disease or medical condition (such as COPD) in a patient,such as a mammal (particularly a human) that includes: (a) preventingthe disease or medical condition from occurring, i.e., prophylactictreatment of a patient; (b) ameliorating the disease or medicalcondition, i.e., eliminating or causing regression of the disease ormedical condition in a patient; (c) suppressing the disease or medicalcondition, i.e., slowing or arresting the development of the disease ormedical condition in a patient; or (d) alleviating the symptoms of thedisease or medical condition in a patient. For example, the term“treating COPD” would include preventing COPD from occurring,ameliorating COPD, suppressing COPD, and alleviating the symptoms ofCOPD. The term “patient” is intended to include those animals, such ashumans, that are in need of treatment or disease prevention, that arepresently being treated for disease prevention or treatment of aspecific disease or medical condition, as well as test subjects in whichcompounds of the invention are being evaluated or being used in a assay,for example an animal model.

All other terms used herein are intended to have their ordinary meaningas understood by those of ordinary skill in the art to which theypertain.

GENERAL SYNTHETIC PROCEDURES

Compounds of the invention can be prepared from readily availablestarting materials using the following general methods, the proceduresset forth in the Examples, or by using other methods, reagents, andstarting materials that are known to those of ordinary skill in the art.Although the following procedures may illustrate a particular embodimentof the invention, it is understood that other embodiments of theinvention can be similarly prepared using the same or similar methods orby using other methods, reagents and starting materials known to thoseof ordinary skill in the art. It will also be appreciated that wheretypical or preferred process conditions (i.e., reaction temperatures,times, mole ratios of reactants, solvents, pressures, etc.) are given,other process conditions can also be used unless otherwise stated. Whileoptimum reaction conditions will typically vary depending on variousreaction parameters such as the particular reactants, solvents andquantities used, those of ordinary skill in the art can readilydetermine suitable reaction conditions using routine optimizationprocedures.

Additionally, as will be apparent to those skilled in the art,conventional protecting groups may be necessary or desired to preventcertain functional groups from undergoing undesired reactions. Thechoice of a suitable protecting group for a particular functional groupas well as suitable conditions and reagents for protection anddeprotection of such functional groups are well-known in the art.Functional groups that may be protected so as to prevent undesiredreactions include, by way of example, carboxy groups, amino groups,hydroxyl groups, thiol groups, carbonyl groups and the like.Representative carboxy-protecting groups include, but are not limitedto, esters, such as methyl, ethyl, tert-butyl, benzyl (Bn),p-methoxybenzyl (PMB), 9-fluoroenylmethyl (Fm), trimethylsilyl (TMS),tert-butyldimethylsilyl (TBS), diphenylmethyl (benzhydryl, DPM) and thelike; amides and hydrazides. Representative protecting groups for aminogroups include carbamates (such as tert-butoxycarbonyl) and amides.Representative hydroxyl-protecting groups include, but are not limitedto, silyl groups including triC₁₋₆alkylsilyl groups, such astrimethylsilyl (TMS), triethylsilyl (TES), tert-butyldimethylsilyl (TBS)and the like; esters (acyl groups) including C₁₋₆alkanoyl groups, suchas formyl, acetyl and the like; arylmethyl groups, such as benzyl (Bn),p-methoxybenzyl (PMB), 9-fluorenylmethyl (Fm), diphenylmethyl(benzhydryl, DPM) and the like; and ethers. Representative protectinggroups for thiol groups include thioethers and thioesters.Representative protecting groups for carbonyl groups include acetals andketals. Protecting groups other than those described herein may be used,if desired. For example, numerous protecting groups, and theirintroduction and removal, are described in T. W. Greene and G. M. Wuts,Protecting Groups in Organic Synthesis, Third Edition, Wiley, New York,1999, and references cited therein.

By way of illustration, compounds of formula I can be prepared by one ormore of the following exemplary processes:

(a) reacting a compound of formula II:

with a compound of formula III:

where L¹ represents a leaving group, to produce a compound of formulaIV:

and reacting the compound of formula IV with an organic substratecontaining an R⁵ group; or

(b) reacting a compound of formula V:

with a compound of formula III; or

(c) reacting a compound of formula V with a compound of formula VI:

where L² represents a leaving group and A is defined below, to produce acompound of formula VII:

and reacting the compound of formula VII with a compound of formulaVIII:

wherein L³ represents a leaving group, and Q′, A and B are defined asset forth in the following table:

Q′ A B —CH₂— —CH₃ L³-alkylene- —CH═CH— H L³-alkenylene- —C≡C— HL³-alkynylene- —O— -L³ HO— —S— -L³ HS— —SO₂—NR^(Q1)— —SO₂—OH or —SO₂ClR^(Q1)HN— —NR^(Q1)—SO₂— —NHR^(Q1) HOO₂S— —OC(O)— —OH HO(O)C— —C(O)O——C(O)OH or —C(O)Cl HO— —NR^(Q1)C(O)— —NHR^(Q1) HO(O)C—or Cl(O)C——NR^(Q2)—C(O)—NR^(Q3)— —N═C═O H₂N— where R^(Q2) and R^(Q3) are H—NR^(Q2)—C(S)—NR^(Q3)— —N═S═O H₂N— where R^(Q2) and R^(Q3) are H —C═N—O——CHO H₂NO— —S—S— —SH HS—and recovering the product in salt or zwitterionic form.

The resulting reaction product, a compound of formula I, is a quaternaryammonium compound, which is readily crystallized in suitable solvents,such as are well known in the art. Such crystals are quaternary ammoniumsalts.

In these reactions, depending upon the particular substituents present,one or more protecting groups may be employed. If such protecting groupsare used, they are removed using conventional procedures to provide thecompound of formula I.

Process (a)

In process (a), the reaction between the compounds of formula II andIII, the leaving group represented by L¹ can be, for example, halo, suchas chloro, bromo or iodo, or a sulfonic ester group, such as mesylate ortosylate. In one embodiment, L¹ is bromo. The reaction is convenientlyperformed in the presence of a base, for example, a tertiary amine suchas diisopropylethylamine. Convenient solvents include nitriles, such asacetonitrile, dimethylformamide (DMF), and dimethylacetamide (DMA). Thereaction is conveniently conducted at a temperature in the range of from0° C. to 100° C. The reaction product is then isolated usingconventional procedures, such as extraction, recrystallization,chromatography and the like.

The compound of formula IV, the free base form of the desired product,is dissolved in a suitable solvent then contacted with an organicsubstrate. Exemplary solvents include toluene, DMA, and CH₃CN. Theorganic substrate is typically a pharmaceutically acceptable acid suchas an organic halide. The substrate contains an R⁵ group, for example,—C₁₋₆alkyl, which may be substituted with 1-5 fluoro atoms and/or 1 to2-O—C₁₋₆alkyl, —OH and phenyl groups, and a leaving group, examples ofwhich include halides such as iodide or bromide. Exemplary substratesinclude methyl iodide, methyl bromide, ethyl iodide, propyl iodide,benzyl bromide and benzyl iodide.

In some situations, process (a) can be followed by a second reaction toyield a different compound of formula I. For example, compounds where Q′is —S(O)— or —SO₂— can be made by forming a compound of formula I whereQ′ is —S—, and subjecting such compound to an oxidation reaction. Inaddition, compounds where Q′ is —C(═N—O—R^(Q4))— can be made by forminga compound of formula I where Q′ is —C(O)—, and subjecting such compoundto an imine formation reaction with H₂N—O—R^(Q4).

Compounds of formula II are generally known in the art or can beprepared from commercially available starting materials and reagentsusing well-known procedures. For example, they may be prepared asdescribed in U.S. Pat. No. 5,096,890 to Cross et al., the disclosure ofwhich is incorporated herein by reference in its entirety.Alternatively, compounds of formula II can be prepared by deprotecting acompound of formula 1:

in which P¹ represents an amino-protecting group, such as a benzylgroup. Benzyl groups are conveniently removed by reduction, for example,using a hydrogen or ammonium formate and a Group VIII metal catalyst,such as palladium. Optionally, this reaction is conducted in thepresence of an acid, such as formic acid, acetic acid, hydrochloricacid, hydrobromic acid, sulfuric acid and the like.

Compounds of formula 1 in which R³ is —C(O)NR^(3a)R^(3b) can be preparedby reacting a carboxylic acid of formula 2:

with an amine of formula HNR^(3a)R^(3b) under amide bond formingconditions, where compounds of formula 2 may be prepared by hydrolyzinga compound of formula 3 using an aqueous solution of a acid, preferablyhydrochloric acid, hydrobromic acid or sulfuric acid:

Compounds of formula 3 can be prepared as described in U.S. Pat. No.5,096,890 to Cross et al.

Compounds of formula 1 in which R³ is —C(O)O—C₁₋₄alkyl can be preparedby reacting a carboxylic acid of formula 2 with a C₁₋₄alkyl alcohol inthe presence of a catalytic amount of an acid, preferably hydrochloricacid, hydrobromic acid or sulfuric acid. Compounds of formula 1 in whichR³ is —CN can be prepared as described in U.S. Pat. No. 5,096,890 toCross et al. Compounds of formula 1 in which R³ is —OH can be preparedby reacting an ester of formula 4 with a nucleophilic source of phenyl;such as phenyl lithium or phenyl magnesium bromide:

Compounds of formula 1 in which R³ is —CH₂OH can be prepared by reactinga carboxylic acid of formula 2 with a reducing agent such as sodiumborohydride, lithium aluminum hydride or borane.

Compounds of formula 1 in which R³ is —CH₂NH₂ can be prepared byreacting a nitrile of formula 3 with a reducing agent such as lithiumaluminum hydride or diisobutyl aluminum hydride.

Compounds of formula III are generally known or can be prepared fromreadily available starting materials using well-known synthetic methods.

Process (b)

In process (b), the reaction between the compounds of formula V and IIIcan be conducted using known procedures for reacting pyrrolidines withhalogenated compounds. The reaction is typically conducted in an organicsolvent at a temperature in the range of from about 20 to 120° C., moretypically in the range of about from about 20 to 80° C. Suitable organicsolvents include acetonitrile, dimethylsulfoxide, dimethylacetamide,ether, and acetone. Compounds of formula V can be prepared by reactingcompounds of formula II with an organic substrate containing an R⁵ groupas described in process (a).

Process (c)

In process (c), the reaction conditions between the compounds of formulaV, VI and VIII will vary depending upon the respective A and B groups.The leaving group represented by L³ can be, for example, a halo,typically bromo. Compounds of formula VI and VIII are generally known orcan be prepared from readily available starting materials usingwell-known synthetic methods.

Some reactions in process (c) are coupling reactions, for example, whenQ′ is —NR^(Q1)C(O)—. In those reactions, the acidic moiety-containingcompound may be in the form of a reactive derivative. For example, thecarboxylic acid may be activated, for example, by forming an anhydrideor carboxylic acid halide, such as a carboxylic acid chloride. Thus thecarboxylic acid chloride is a reactive derivative of carboxylic acid.Alternatively, the carboxylic acid can be activated using conventionalcarboxylic acid/amine coupling reagents, such carbodiimides,O-(7-azabenzotriazol-1-yl-N,N,N′,N′ tetramethyluroniumhexafluorophosphate (HATU) and the like. The sulfonic acid and thio acidmoieties can be similarly derivatized. The reactions are conducted underconventional conditions using suitable coupling agents such ascarbonyldiimidazole. The reaction is typically conducted in the presenceof solvents such as trifluoroacetic acid and dichloromethane, andconveniently conducted at a temperature in the range of from −10° C. to100° C.

The remaining reactions in process (c) are alkylation reactions, forexample, when Q′ is —O—. The reactions are conducted under conventionalconditions using suitable solvents such as DMF or DMA, and convenientlyconducted at a temperature in the range of from room temperature to 100°C.

In addition, process (c) illustrates formation compounds of formula Iwhere the R^(Q2) and/or R^(Q3) moieties are hydrogen. Such compounds arereadily converted to compounds of formula 1 where R^(Q2) and/or R^(Q3)are a C₁₋₄alkyl or C₃₋₆cycloalkyl moiety, or are taken together to forman alkylene or alkenylene linkage.

Further details regarding specific reaction conditions and otherprocedures for preparing representative compounds of the invention orintermediates thereof are described in the Examples set forth below.

Pharmaceutical Compositions and Formulations

Compounds of the invention are typically administered to a patient inthe form of a pharmaceutical composition or formulation. Suchpharmaceutical compositions may be administered to the patient by anyacceptable route of administration including, but not limited to,inhaled, oral, nasal, topical (including transdermal) and parenteralmodes of administration. Further, the compounds of the invention may beadministered, for example orally, in multiple doses per day, in a singledaily dose or a single weekly dose. It will be understood that any formof the compounds of the invention, (i.e., free base, pharmaceuticallyacceptable salt, solvate, etc.) that is suitable for the particular modeof administration can be used in the pharmaceutical compositionsdiscussed herein.

Accordingly, in one embodiment, the invention is directed to apharmaceutical composition comprising a pharmaceutically acceptablecarrier and a compound of the invention. The compositions may containother therapeutic and/or formulating agents if desired. A “compound ofthe invention” may also be referred to herein as the “active agent.”

The pharmaceutical compositions of this invention typically contain atherapeutically effective amount of a compound of the invention. Thoseskilled in the art will recognize, however, that a pharmaceuticalcomposition may contain more than a therapeutically effective amount,i.e., bulk compositions, or less than a therapeutically effectiveamount, i.e., individual unit doses designed for multiple administrationto achieve a therapeutically effective amount. In one embodiment, thecomposition will contain from about 0.01-95 wt % of active agent,including, from about 0.01-30 wt %, such as from about 0.01-10 wt %,with the actual amount depending upon the formulation itself, the routeof administration, the frequency of dosing, and so forth. In anotherembodiment, a composition suitable for inhalation, for example,comprises from about 0.01-30 wt % or active agent with yet anotherembodiment comprises from about 0.01-10 wt % active agent.

Any conventional carrier or excipient may be used in the pharmaceuticalcompositions of the invention. The choice of a particular carrier orexcipient, or combinations of carriers or excipients, will depend on themode of administration being used to treat a particular patient or typeof medical condition or disease state. In this regard, the preparationof a suitable composition for a particular mode of administration iswell within the scope of those skilled in the pharmaceutical arts.Additionally, carriers or excipients used in such compositions arecommercially available. By way of further illustration, conventionalformulation techniques are described in Remington: The Science andPractice of Pharmacy, 20^(th) Edition, Lippincott Williams & White,Baltimore, Md. (2000); and H. C. Ansel et al., Pharmaceutical DosageForms and Drug Delivery Systems, 7^(th) Edition, Lippincott Williams &White, Baltimore, Md. (1999).

Representative examples of materials which can serve as pharmaceuticallyacceptable carriers include, but are not limited to, the following:sugars, such as lactose, glucose and sucrose; starches, such as cornstarch and potato starch; cellulose, such as microcrystalline cellulose,and its derivatives, such as sodium carboxymethyl cellulose, ethylcellulose and cellulose acetate; powdered tragacanth; malt; gelatin;talc; excipients, such as cocoa butter and suppository waxes; oils, suchas peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil,corn oil and soybean oil; glycols, such as propylene glycol; polyols,such as glycerin, sorbitol, mannitol and polyethylene glycol; esters,such as ethyl oleate and ethyl laurate; agar; buffering agents, such asmagnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-freewater; isotonic saline; Ringer's solution; ethyl alcohol; phosphatebuffer solutions; compressed propellant gases, such aschlorofluorocarbons and hydrofluorocarbons; and other non-toxiccompatible substances employed in pharmaceutical compositions.

Pharmaceutical compositions are typically prepared by thoroughly andintimately mixing or blending the active agent with a pharmaceuticallyacceptable carrier and one or more optional ingredients. The resultinguniformly blended mixture may then be shaped or loaded into tablets,capsules, pills, canisters, cartridges, dispensers and the like usingconventional procedures and equipment.

In one embodiment, the pharmaceutical compositions are suitable forinhaled administration. Suitable compositions for inhaled administrationwill typically be in the form of an aerosol or a powder. Suchcompositions are generally administered using well-known deliverydevices, such as a nebulizer inhaler, a dry powder inhaler, or ametered-dose inhaler, examples of which are described below.

In a specific embodiment of the invention, a composition comprising theactive agent is administered by inhalation using a nebulizer inhaler.Such nebulizer devices typically produce a stream of high velocity airthat causes the composition to spray as a mist that is carried into apatient's respiratory tract. Accordingly, when formulated for use in anebulizer inhaler, the active agent is typically dissolved in a suitablecarrier to form a solution. Alternatively, the active agent can bemicronized and combined with a suitable carrier to form a suspension ofmicronized particles of respirable size, where micronized is typicallydefined as having particles in which at least about 90 percent of theparticles have a mass median diameter of less than about 10 μm. The term“mass median diameter” means the diameter such that half the mass of theparticles is contained in particles with larger diameter and half iscontained in particles with smaller diameter.

Suitable nebulizer devices include the Respimat® Soft Mist™ Inhaler(Boehringer Ingelheim), the AERx® Pulmonary Delivery System (AradigmCorp.), and the PARI LC Plus Reusable Nebulizer (Pari GmbH). Anexemplary composition for use in a nebulizer inhaler comprises anisotonic aqueous solution comprising from about 0.05 μg/mL to about 10mg/mL of a compound of the invention. In one embodiment, such a solutionhas a pH of about 4-6.

In another specific embodiment of the invention, a compositioncomprising the active agent is administered by inhalation using a drypowder inhaler (DPI). Such DPIs typically administer the active agent asa free-flowing powder that is dispersed in a patient's air-stream duringinspiration. In order to achieve a free flowing powder, the active agentis typically formulated with a suitable excipient such as lactose,starch, mannitol, dextrose, polylactic acid, polylactide-co-glycolide,and combinations thereof. Typically, the active agent is micronized andcombined with an excipient to form a blend suitable for inhalation.Accordingly, in one embodiment of the invention, the active agent is inmicronized form. For example, a representative composition for use in aDPI comprises dry lactose having a particle size between about 1 μm andabout 100 μm (e.g., dry milled lactose) and micronized particles of theactive agent. Such a dry powder formulation can be made, for example, bycombining lactose with the active agent and then dry blending thecomponents. Alternatively, if desired, the active agent can beformulated without an excipient. The composition is then typicallyloaded into a DPI, or into inhalation cartridges or capsules for usewith a DPI. DPIs are well known to those of ordinary skill in the art,and many such devices are commercially available, with representativedevices including Aerolizer® (Novartis), Airmax™ (IVAX), ClickHaler®(Innovata Biomed), Diskhaler® (GlaxoSmithKline), Diskus® or Accuhaler(GlaxoSmithKline), Easyhaler® (Orion Pharma), Eclipse™ (Aventis),FlowCaps® (Hovione), Handihaler® (Boehringer Ingelheim), Pulvinal®(Chiesi), Rotahaler® (GlaxoSmithKline), SkyeHaler™ or Certihaler™(SkyePharma), Twisthaler (Schering-Plough), Turbuhaler® (AstraZeneca),Ultrahaler® (Aventis), and the like.

In yet another specific embodiment of the invention, the compositioncomprising the active agent is administered by inhalation using ametered-dose inhaler (MDI). Such MDIs typically discharge a measuredamount of the active agent using compressed propellant gas. Metered-doseformulations thus typically comprise a solution or suspension of theactive agent in a liquefied propellant, such as a chlorofluorocarbonsuch as CCl₃F or a hydrofluoroalkane (HFA) such as1,1,1,2-tetrafluoroethane (HFA 134a) and1,1,1,2,3,3,3-heptafluoro-n-propane (HFA 227), although HFAs aregenerally preferred due to concerns about chlorofluorocarbons affectingthe ozone layer. Additional optional components of HFA formulationsinclude co-solvents, such as ethanol or pentane, and surfactants, suchas sorbitan trioleate, oleic acid, lecithin, and glycerin. See, forexample, U.S. Pat. No. 5,225,183 to Purewal et al., EP 0717987 A2(Minnesota Mining and Manufacturing Company), and WO 92/22286 (MinnesotaMining and Manufacturing Company). A representative composition for usein an MDI comprises from about 0.01-5 wt % of active agent; from about0-20 wt % ethanol; and from about 0-5 wt % surfactant; with theremainder being an HFA propellant. Such compositions are typicallyprepared by adding a chilled or pressurized hydrofluoroalkane to asuitable container containing the active agent, ethanol (if present) andthe surfactant (if present). To prepare a suspension, the active agentis micronized and then combined with the propellant. The formulation isthen loaded into an aerosol canister, which forms a portion of the MDI.MDIs are well known to those of ordinary skill in the art, and many suchdevices are commercially available, with representative devicesincluding AeroBid Inhaler System (Forest Pharmaceuticals), AtroventInhalation Aerosol (Boehringer Ingelheim), Flovent® (GlaxoSmithKline),Maxair Inhaler (3M), Proventil® Inhaler (Schering), Serevent® InhalationAerosol (GlaxoSmithKline), and the like. Alternatively, a suspensionformulation can be prepared by spray drying a coating of surfactant onmicronized particles of the active agent. See, for example, WO 99/53901(Glaxo Group Ltd.) and WO 00/61108 (Glaxo Group Ltd.).

Additional examples of processes of preparing respirable particles, andformulations and devices suitable for inhalation dosing are described inU.S. Pat. No. 5,874,063 to Briggner et al.; U.S. Pat. No. 5,983,956 toTrofast; U.S. Pat. No. 6,221,398 to Jakupovic et al.; U.S. Pat. No.6,268,533 to Gao et al.; U.S. Pat. No. 6,475,524 to Bisrat et al.; andU.S. Pat. No. 6,613,307 to Cooper.

In another embodiment, the pharmaceutical compositions are suitable fororal administration. Suitable compositions for oral administration maybe in the form of capsules, tablets, pills, lozenges, cachets, dragees,powders, granules; solutions or suspensions in an aqueous or non-aqueousliquid; oil-in-water or water-in-oil liquid emulsions; elixirs orsyrups; and the like; each containing a predetermined amount of theactive agent.

When intended for oral administration in a solid dosage form (i.e., ascapsules, tablets, pills and the like), the composition will typicallycomprise the active agent and one or more pharmaceutically acceptablecarriers, such as sodium citrate or dicalcium phosphate. Solid dosageforms may also comprise: fillers or extenders, such as starches,microcrystalline cellulose, lactose, sucrose, glucose, mannitol, and/orsilicic acid; binders, such as carboxymethylcellulose, alginates,gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; humectants, suchas glycerol; disintegrating agents, such as agar-agar, calciumcarbonate, potato or tapioca starch, alginic acid, certain silicates,and/or sodium carbonate; solution retarding agents, such as paraffin;absorption accelerators, such as quaternary ammonium compounds; wettingagents, such as cetyl alcohol and/or glycerol monostearate; absorbents,such as kaolin and/or bentonite clay; lubricants, such as talc, calciumstearate, magnesium stearate, solid polyethylene glycols, sodium laurylsulfate, and/or mixtures thereof; coloring agents; and buffering agents.

Release agents, wetting agents, coating agents, sweetening, flavoringand perfuming agents, preservatives and antioxidants may also be presentin the pharmaceutical compositions. Exemplary coating agents fortablets, capsules, pills and like, include those used for entericcoatings, such as cellulose acetate phthalate, polyvinyl acetatephthalate, hydroxypropyl methylcellulose phthalate, methacrylicacid-methacrylic acid ester copolymers, cellulose acetate trimellitate,carboxymethyl ethyl cellulose, hydroxypropyl methyl cellulose acetatesuccinate, and the like. Examples of pharmaceutically acceptableantioxidants include: water-soluble antioxidants, such as ascorbic acid,cysteine hydrochloride, sodium bisulfate, sodium metabisulfate sodiumsulfite and the like; oil-soluble antioxidants, such as ascorbylpalmitate, butylated hydroxyanisole, butylated hydroxytoluene, lecithin,propyl gallate, alpha-tocopherol, and the like; and metal-chelatingagents, such as citric acid, ethylenediamine tetraacetic acid, sorbitol,tartaric acid, phosphoric acid, and the like.

Compositions may also be formulated to provide slow or controlledrelease of the active agent using, by way of example, hydroxypropylmethyl cellulose in varying proportions or other polymer matrices,liposomes and/or microspheres. In addition, the pharmaceuticalcompositions of the invention may contain opacifying agents and may beformulated so that they release the active agent only, orpreferentially, in a certain portion of the gastrointestinal tract,optionally, in a delayed manner. Examples of embedding compositionswhich can be used include polymeric substances and waxes. The activeagent can also be in micro-encapsulated form, if appropriate, with oneor more of the above-described excipients.

Suitable liquid dosage forms for oral administration include, by way ofillustration, pharmaceutically acceptable emulsions, microemulsions,solutions, suspensions, syrups and elixirs. Liquid dosage formstypically comprise the active agent and an inert diluent, such as, forexample, water or other solvents, solubilizing agents and emulsifiers,such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethylacetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyleneglycol, oils (e.g., cottonseed, groundnut, corn, germ, olive, castor andsesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycolsand fatty acid esters of sorbitan, and mixtures thereof. Suspensions maycontain suspending agents such as, for example, ethoxylated isostearylalcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystallinecellulose, aluminium metahydroxide, bentonite, agar-agar and tragacanth,and mixtures thereof.

When intended for oral administration, the pharmaceutical compositionsof the invention may be packaged in a unit dosage form. The term “unitdosage form” refers to a physically discrete unit suitable for dosing apatient, i.e., each unit containing a predetermined quantity of theactive agent calculated to produce the desired therapeutic effect eitheralone or in combination with one or more additional units. For example,such unit dosage forms may be capsules, tablets, pills, and the like.

Compounds of the invention can also be administered parenterally (e.g.,by subcutaneous, intravenous, intramuscular, or intraperitonealinjection). For such administration, the active agent is provided in asterile solution, suspension, or emulsion. Exemplary solvents forpreparing such formulations include water, saline, low molecular weightalcohols such as propylene glycol, polyethylene glycol, oils, gelatin,fatty acid esters such as ethyl oleate, and the like. A typicalparenteral formulation is a sterile pH 4-7 aqueous solution of theactive agent. Parenteral formulations may also contain one or moresolubilizers, stabilizers, preservatives, wetting agents, emulsifiers,and dispersing agents. These formulations may be rendered sterile by useof a sterile injectable medium, a sterilizing agent, filtration,irradiation, or heat.

Compounds of the invention can also be administered transdermally usingknown transdermal delivery systems and excipients. For example, thecompound can be admixed with permeation enhancers, such as propyleneglycol, polyethylene glycol monolaurate, azacycloalkan-2-ones and thelike, and incorporated into a patch or similar delivery system.Additional excipients including gelling agents, emulsifiers and buffers,may be used in such transdermal compositions if desired.

If desired, the compounds of this invention may be administered incombination with one or more other therapeutic agents. Thus, in oneembodiment, compositions of the invention may optionally contain otherdrugs that are co-administered with a compound of the invention. Forexample, the composition may further comprise one or more drugs (alsoreferred to as “secondary agents(s)”) selected from the group of otherbronchodilators (e.g., PDE₃ inhibitors, adenosine 2b modulators and β₂adrenergic receptor agonists); anti-inflammatory agents (e.g., steroidalanti-inflammatory agents such as corticosteroids and glucocorticoids;non-steroidal anti-inflammatory agents (NSAIDs); and PDE₄ inhibitors);other muscarinic receptor antagonists (i.e., antichlolinergic agents);antiinfective agents (e.g., Gram positive and Gram negative antibiotics,and antiviral agents); antihistamines; protease inhibitors; afferentblockers (e.g., D₂ agonists and neurokinin modulators); and combinationsthereof. Numerous examples of such therapeutic agents are well known inthe art, and examples are described below. By combining a compound ofthe invention with a secondary agent, double therapy can be achieved,i.e., muscarinic receptor antagonist activity and activity associatedwith the secondary agent (e.g., β₁ adrenergic receptor agonist), in somecases by administering two compositions and in some cases byadministering a single composition containing the active agent and thesecondary agent. Accordingly, in yet another aspect of the invention, apharmaceutical composition comprises a compound of the invention, asecond active agent, and a pharmaceutically acceptable carrier. Third,fourth etc. active agents may also be included in the composition. Forexample, a composition may comprise a compound of the invention; asecondary agent selected from corticosteroids, β₂ adrenergic receptoragonists; phosphodiesterase-4 inhibitors, and combinations thereof; anda pharmaceutically acceptable carrier. In a specific embodiment, thecomposition comprises a compound of the invention, a β₂ adrenergicreceptor agonist, and a steroidal anti-inflammatory agent. Incombination therapy, the amount of compound of the invention that isadministered, as well as the amount of secondary agents, may be lessthan the amount typically administered in monotherapy.

A compound of the invention may be either physically mixed with thesecond active agent to form a composition containing both agents; oreach agent may be present in separate and distinct compositions whichare administered to the patient simultaneously or sequentially. Forexample, a compound of the invention can be combined with a secondactive agent using conventional procedures and equipment to form acombination of active agents comprising a compound of the invention anda second active agent. Additionally, the active agents may be combinedwith a pharmaceutically acceptable carrier to form a pharmaceuticalcomposition comprising a compound of the invention, a second activeagent and a pharmaceutically acceptable carrier. In this embodiment, thecomponents of the composition are typically mixed or blended to create aphysical mixture. The physical mixture is then administered in atherapeutically effective amount using any of the routes describedherein.

Alternatively, the active agents may remain separate and distinct beforeadministration to the patient. In this embodiment, the agents are notphysically mixed together before administration but are administeredsimultaneously or at separate times as separate compositions. Suchcompositions can be packaged separately or may be packaged together in akit. When administered at separate times, the secondary agent willtypically be administered less than 24 hours after administration of thecompound of the invention. In other embodiments this timed relationshipis less than 12 hours, less than 8 hours, less than 6 hours, less than 4hours, less than 3 hours, less than 1 hour, less than thirty minutes,less than ten minutes, less than one minute, or immediately afteradministration of the compound of the invention. This is also referredto as sequential administration. Thus, a compound of the invention canbe administered by inhalation simultaneously or sequentially withanother active agent using an inhalation delivery device that employsseparate compartments (e.g. blister packs) for each active agent, wheresequential may mean being administered immediately after administrationof the compound of the invention or at some predetermined time later(e.g., one hour later or three hours later).

Alternatively, the combination may be administered using separatedelivery devices, i.e., one delivery device for each agent.Additionally, the agents can be delivered by different routes ofadministration, i.e., one by inhalation and the other by oraladministration. In one embodiment, the kit comprises a first dosage formcomprising a compound of the invention and at least one additionaldosage form comprising one or more of the secondary agents set forthherein, in quantities sufficient to carry out the methods of theinvention. The first dosage form and the second (or third, etc,) dosageform together comprise a therapeutically effective amount of activeagents for the treatment or prevention of a disease or medical conditionin a patient.

Secondary agent(s), when included, are present in a therapeuticallyeffective amount. i.e., are typically administered in an amount thatproduces a therapeutically beneficial effect when co-administered with acompound of the invention. The secondary agent can be in the form of apharmaceutically acceptable salt, solvate, optically pure stereoisomer,and so forth. Thus, secondary agents listed below are intended toinclude all such forms, and are commercially available or can beprepared using conventional procedures and reagents. Suitable doses fora secondary agent are typically in the range of about 0.05 μg/day toabout 500 mg/day.

In a particular embodiment, a compound of the invention is administeredin combination with a β₂ adrenergic receptor agonist. Representative β₂adrenergic receptor agonists include, but are not limited to, albuterol,bitolterol, fenoterol, formoterol, indacaterol, isoetharine,levalbuterol, metaproterenol, pirbuterol, salbutamol, salmefamol,salmeterol, terbutaline, and the like. Other β₂ adrenergic receptoragonists that can be used in combination with compounds of the inventioninclude, but are not limited to,3-(4-{[6-({(2R)-2-hydroxy-2-[4-hydroxy-3-(hydroxymethyl)-phenyl]ethyl}amino)-hexyl]oxy}butyl)benzenesulfonamideand3-(−3-{[7-({(2R)-2-hydroxy-2-[4-hydroxy-3-(hydroxymethyl)phenyl]ethyl}-amino)heptyl]oxy}-propyl)benzenesulfonamideand related compounds disclosed in WO 02/066422 (Glaxo Group Ltd.);3-[3-(4-{[6-([(2R)-2-hydroxy-2-[4-hydroxy-3-(hydroxymethyl)phenyl]ethyl}amino)hexyl]oxy}butyl)-phenyl]imidazolidine-2,4-dioneand related compounds disclosed in WO 02/070490 (Glaxo Group Ltd.);3-(4-{[6-({(2R)-2-[3-(formylamino)-4-hydroxyphenyl]-2-hydroxyethyl}amino)hexyl]oxy}butyl)-benzenesulfonamide,3-(4-{[6-({(2S)-2-[3-(formylamino)-4-hydroxyphenyl]-2-hydroxyethyl}amino)hexyl]oxy}butyl)-benzenesulfonamide,3-(4-{[6-({(2R/S)-2-[3-(formylamino)-4-hydroxyphenyl]-2-hydroxyethyl}amino)hexyl]oxy}butyl)-benzenesulfonamide,N-(tert-butyl)-3-(4-{[6-({(2R)-2-[3-(formylamino)-4-hydroxyphenyl]-2-hydroxyethyl}amino)hexyl]-oxy}butyl)benzenesulfonamide,N-(tert-butyl)-3-(4-{[6-({(2S)-2-[3-(formylamino)-4-hydroxyphenyl]-2-hydroxyethyl}amino)-hexyl]oxy}butyl)-benzenesulfonamide,N-(tert-butyl)-3-(4-{[6-({(2R/S)-2-[3-(formylamino)-4-hydroxyphenyl]-2-hydroxyethyl}amino)hexyl]-oxy}butyl)benzenesulfonamide and related compounds disclosed inWO 02/076933 (Glaxo Group Ltd.);4-{(1R)-2-[(6-{2-[(2,6-dichlorobenzyl)oxy]ethoxy}hexyl)amino]-1-hydroxyethyl}-2-(hydroxymethyl)phenoland related compounds disclosed in WO 03/024439 (Glaxo Group Ltd.);N-{2-[4-((R)-2-hydroxy-2-phenylethylamino)phenyl]ethyl}-(R)-2-hydroxy-2-(3-formamido-4-hydroxyphenyl)ethylamineand related compounds disclosed in U.S. Pat. No. 6,576,793 to Moran etal.;N-{2-[4-(3-phenyl-4-methoxyphenyl)aminophenyl]ethyl}-(R)-2-hydroxy-2-(8-hydroxy-2(1H)-quinolinon-5-yl)ethylamineand related compounds disclosed in U.S. Pat. No. 6,653,323 to Moran etal. In a particular embodiment, the β₂-adrenoreceptor agonist is acrystalline monohydrochloride salt ofN-{2-[4-((R)-2-hydroxy-2-phenylethylamino)phenyl]ethyl}-(R)-2-hydroxy-2-(3-formamido-4-hydroxyphenyl)ethylamine.Typically, the β₂-adrenoreceptor agonist will be administered in anamount sufficient to provide from about 0.05-500 μg per dose.

In a particular embodiment, a compound of the invention is administeredin combination with a steroidal anti-inflammatory agent. Representativesteroidal anti-inflammatory agents include, but are not limited to,beclomethasone dipropionate; budesonide; butixocort propionate;20R-16α,17α-[butylidenebis(oxy)]-6α,9α-difluoro-1,3-hydroxy-17β-(methylthio)androsta-4-en-3-one(RPR-106541); ciclesonide; dexamethasone;6α,9α-difluoro-17α-[(2-furanylcarbonyl)oxy]-11β-hydroxy-16α-methyl-3-oxoandrosta-1,4-diene-17β-carbothioicacid S-fluoromethyl ester;6α,9α-difluoro-1,3-hydroxy-16α-methyl-17α-[(4-methyl-11β-thiazole-5-carbonyl)oxy]-3-oxoandrosta-1,4-diene-17β-carbothioicacid S-fluoromethyl ester;6α,9α-difluoro-11β-hydroxy-16α-methyl-3-oxo-17α-propionyloxyandrosta-1,4-diene-17β-carbothioicacid (S)-(2-oxotetrahydrofuran-3S-yl) ester; flunisolide; fluticasonepropionate; methyl prednisolone; mometasone furoate; prednisolone;prednisone; rofleponide; ST-126; triamcinolone acetonide; and the like.Typically, the steroidal anti-inflammatory agent will be administered inan amount sufficient to provide from about 0.05-500 g per dose.

An exemplary combination is a compound of the invention co-administeredwith salmeterol as the β₂ adrenergic receptor agonist, and fluticasonepropionate as the steroidal anti-inflammatory agent. Another exemplarycombination is a compound of the invention co-administered with acrystalline monohydrochloride salt ofN-({2-[4-((R)-2-hydroxy-2-phenylethylamino)phenyl]ethyl})-(R)-2-hydroxy-2-(3-formamido-4-hydroxyphenyl)ethylamineas the β₂-adrenoreceptor agonist, and6α,9α-difluoro-17α-[(2-furanylcarbonyl)oxy]-11β-hydroxy-16α-methyl-3-oxoandrosta-1,4-diene-17β-carbothioicacid S-fluoromethyl ester as the steroidal anti-inflammatory agent.

Other suitable combinations include, for example, otheranti-inflammatory agents, e.g., NSAIDs (such as sodium cromoglycate;nedocromil sodium; phosphodiesterase (PDE) inhibitors (e.g.,theophylline, PDE4 inhibitors or mixed PDE3/PDE4 inhibitors);leukotriene antagonists (e.g., monteleukast); inhibitors of leukotrienesynthesis; iNOS inhibitors; protease inhibitors, such as tryptase andelastase inhibitors; beta-2 integrin antagonists and adenosine receptoragonists or antagonists (e.g., adenosine 2a agonists); cytokineantagonists (e.g., chemokine antagonists such as, an interleukinantibody (αIL antibody), specifically, an αIL-4 therapy, an αIL-13therapy, or a combination thereof); or inhibitors of cytokine synthesis.

In a particular embodiment, a compound of the invention is administeredin combination with a phosphodiesterase-4 (PDE4) inhibitors or mixedPDE3/PDE4 inhibitors. Representative PDE4 or mixed PDE3/PDE4 inhibitorsinclude, but are not limited to, c is4-cyano-4-(3-cyclopentyloxy-4-methoxyphenyl)cyclohexan-1-carboxylicacid,2-carbomethoxy-4-cyano-4-(3-cyclopropylmethoxy-4-difluoromethoxyphenyl)cyclohexan-1-one;cis-[4-cyano-4-(3-cyclopropylmethoxy-4-difluoromethoxyphenyl)cyclohexan-1-ol];cis-4-cyano-4-[3-(cyclopentyloxy)-4-methoxyphenyl]cyclohexane-1-carboxylicacid and the like, or pharmaceutically acceptable salts thereof. Otherrepresentative PDE4 or mixed PDE4/PDE3 inhibitors include AWD-12-281(elbion); NCS-613 (INSERM); D-4418 (Chiroscience and Schering-Plough);CI-1018 or PD-168787 (Pfizer); benzodioxole compounds disclosed inWO99/16766 (Kyowa Hakko); K-34 (Kyowa Hakko); V-11294A (Napp);roflumilast (Byk-Gulden); pthalazinone compounds disclosed in WO99/47505(Byk-Gulden); Pumafentrine (Byk-Gulden, now Altana); arofylline(Almirall-Prodesfarma); VM554/UM565 (Vernalis); T-440 (Tanabe Seiyaku);and T2585 (Tanabe Seiyaku).

In a particular embodiment, a compound of the invention is administeredin combination with a muscarinic antagonist (i.e., anticholinergicagent). Representative muscarinic antagonists include, but are notlimited to, atropine, atropine sulfate, atropine oxide, methylatropinenitrate, homatropine hydrobromide, hyoscyamine (d, l) hydrobromide,scopolamine hydrobromide, ipratropium bromide, oxitropium bromide,tiotropium bromriide, methantheline, propantheline bromide, anisotropinemethyl bromide, clidinium bromide, copyrrolate (Robinul), isopropamideiodide, mepenzolate bromide, tridihexethyl chloride (Pathilone),hexocyclium methylsulfate, cyclopentolate hydrochloride, tropicamide,trihexyphenidyl hydrochloride, pirenzepine, telenzepine, AF-DX 116 andmethoctramine and the like.

In a particular embodiment, a compound of the invention is administeredin combination with an antihistamine (i.e., H₁-receptor antagonist).Representative antihistamines include, but are not limited to,ethanolamines, such as carbinoxamine maleate, clemastine fumarate,diphenylhydramine hydrochloride and dimenhydrinate; ethylenediamines,such as pyrilamine amleate, tripelennamine hydrochloride andtripelennamine citrate; alkylamines, such as chlorpheniramine andacrivastine; piperazines, such as hydroxyzine hydrochloride, hydroxyzinepamoate, cyclizine hydrochloride, cyclizine lactate, meclizinehydrochloride and cetirizine hydrochloride; piperidines, such asastemizole, levocabastine hydrochloride, loratadine or itsdescarboethoxy analogue, terfenadine and fexofenadine hydrochloride;azelastine hydrochloride; and the like.

The following formulations illustrate representative pharmaceuticalcompositions of the invention.

Exemplary Compositions for Administration by a DPI

A compound of the invention (0.2 mg) is micronized and then blended withlactose (25 mg). This blended mixture is then loaded into a gelatininhalation cartridge. The contents of the cartridge are administeredusing a DPI, for example.

A micronized compound of the invention (100 mg) is blended with milledlactose (25 g) (e.g., lactose in which not greater than about 85% of theparticles have a MMD of about 60 μm to about 90 μm and not less than 15%of the particles have a MMD of less then 15 μm). The blended mixture isthen loaded into individual blisters of a peelable blister pack in anamount sufficient to provide about 10-500 μg of the compound of theinvention per dose. The contents of the blisters are administered usinga DPI.

Alternately, a micronized compound of the invention (1 g) is blendedwith milled lactose (200 g) to form a bulk composition having a weightratio of compound to milled lactose of 1:200. The blended composition ispacked into a DPI capable of delivering between about 10-500 μg of thecompound of the invention per dose.

Alternately, a micronized compound of the invention (100 mg) and amicronized β₂ adrenergic receptor agonist (500 mg) are blended withmilled lactose (30 g). The blended mixture is then loaded intoindividual blisters of a peelable blister pack in an amount sufficientto provide about 10 μg to about 500 μg of the compound of the inventionper dose. The contents of the blisters are administered using a DPI.

Exemplary Compositions for Use in an MDI

A micronized compound of the invention (10 g) is dispersed in a solutionprepared by dissolving lecithin (0.2 g) in demineralized water (200 mL).The resulting suspension is spray dried and then micronized to form amicronized composition comprising particles having a mean diameter lessthan about 1.5 μm. The micronized composition is then loaded into MDIcartridges containing pressurized 1,1,1,2-tetrafluoroethane in an amountsufficient to provide about 10 μg to about 500 μg of the compound of theinvention per dose when administered by the MDI.

Alternately, a suspension containing 5 wt % compound of the invention,0.5 wt % lecithin, and 0.5 wt % trehalose is prepared by dispersing 5 gof a compound of the invention as micronized particles with mean sizeless than 10 μm in a colloidal solution formed from 0.5 g of trehaloseand 0.5 g of lecithin dissolved in 100 mL of demineralized water. Thesuspension is spray dried and the resulting material is micronized toparticles having a mean diameter less than 1.5 μm. The particles areloaded into canisters with pressurized 1,1,1,2-tetrafluoroethane.

Exemplary Composition for Use in a Nebulizer Inhaler

A compound of the invention (25 mg) is dissolved in citrate buffered (pH5) isotonic saline (125 mL). The mixture is stirred and sonicated untilthe compound is dissolved. The pH of the solution is checked andadjusted, if necessary, to pH 5 by slowly adding aqueous 1N sodiumhydroxide. The solution is administered using a nebulizer device thatprovides about 10 μg to about 500 μg of the compound of the inventionper dose.

Exemplary Hard Gelatin Capsules for Oral Administration

A compound of the invention (50 g), spray-dried lactose (440 g) andmagnesium stearate (10 g) are thoroughly blended. The resultingcomposition is then loaded into hard gelatin capsules (500 mg ofcomposition per capsule).

Exemplary Suspension for Oral Administration

The following ingredients are mixed to form a suspension containing 100mg of compound per 10 mL of suspension:

Ingredients Amount Compound of the invention 1.0 g Fumaric acid 0.5 gSodium chloride 2.0 g Methyl paraben 0.15 g Propyl paraben 0.05 gGranulated sugar 25.5 g Sorbitol (70% solution) 12.85 g Veegum ® K(magnesium aluminum silicate) 1.0 g Flavoring 0.035 mL Colorings 0.5 mgDistilled water q.s. to 100 mL

Exemplary Injectable Formulation for Administration by Injection

compound of the invention (0.2 g) is blended with 0.4 M sodium acetatebuffer solution (2.0 mL). The pH of the resulting solution is adjustedto pH 4 using 0.5 N aqueous hydrochloric acid or 0.5 N aqueous sodiumhydroxide, as necessary, and then sufficient water for injection isadded to provide a total volume of 20 mL. The mixture is then filteredthrough a sterile filter (0.22 micron) to provide a sterile solutionsuitable for administration by injection.

UTILITY

Compounds of the invention possess muscarinic receptor antagonistactivity, and in one embodiment, at nanomolar potencies. In oneembodiment, compounds of the invention are selective for inhibition ofM₃ muscarinic receptor subtype activity over M₂ muscarinic receptorsubtype activity. In another embodiment, compounds of the invention areselective for inhibition of M₃ and M₂ muscarinic receptor subtypeactivity over M₁, M₄, and M₅ muscarinic receptor subtype activity.Additionally, compounds of the invention are expected to possess adesirable duration of action. Accordingly, in another specificembodiment, the invention is directed to compounds having a duration ofaction greater than about 24 hours. Moreover, compounds of the inventionare also expected to possess reduced side effects, such as dry mouth, atefficacious doses when administered by inhalation compared to otherknown muscarinic receptor antagonists administered by inhalation (suchas tiotropium).

One measure of the affinity of a compound for the M₃ receptor subtype isthe inhibition dissociation constant (K_(i)) for binding to thereceptor. Compounds of the invention are expected to have a K_(i) forthe M₃ receptor subtype of less than or equal to 100 nM, as determined,for example, by an in vitro radioligand displacement assay. Compounds ofparticular interest include those having a K_(i) less than or equal to50 nM, and in another embodiment, the compounds have a K_(i) less thanor equal to 10 nM, and in yet another embodiment, the compounds have aK_(i) less than or equal to 1.0 nM. Compounds of even more particularinterest include those having a K_(i) less than or equal to 500 pM, andin another embodiment, the compounds have a K_(i) less than or equal to200 pM. It is noted that in some cases, compounds of the invention maypossess weak muscarinic receptor antagonist activity. In such cases,those of skill in the art will recognize that these compounds still haveutility as research tools.

Also of particular interest are those compounds having an ID₅₀ of lessthan or equal to 100 μg/mL at 24 hours post dosing, more particularlythose compounds having an ID₅₀ of less than or equal to 30 μg/mL at 24hours post dosing.

Exemplary assays to determine properties of compounds of the invention,such as the muscarinic receptor antagonizing activity, are described inthe Examples and include by way of illustration and not limitation,assays that measure hM₁, hM₂, hM₃, hM₄, and hM₅ muscarinic receptorbinding (for example, as described in Assay 1). Useful functional assaysto determine the muscarinic receptor antagonizing activity of compoundsof the invention include by way of illustration and not limitation,assays that measure ligand-mediated changes in intracellular cyclicadenosine monophosphate (cAMP), ligand-mediated changes in activity ofthe enzyme adenylyl cyclase (which synthesizes cAMP), ligand-mediatedchanges in incorporation of guanosine 5′-O-(γ-thio)triphosphate([³⁵S]GTPγS) into isolated membranes via receptor catalyzed exchange of[³⁵S]GTPγS for GDP, ligand-mediated changes in free intracellularcalcium ions (measured, for example, with a fluorescence-linked imagingplate reader or FLIPR® from Molecular Devices, Inc.), and the like.Exemplary assays are described in Assay 2. Compounds of this inventionare expected to antagonize or decrease the activation of muscarinicreceptors in any of the assays listed above, or assays of a similarnature, and will typically be used in these studies at a concentrationranging from about 0.1-100 nanomolar. Thus, the aforementioned assaysare useful in determining the therapeutic utility, for example, thebronchodilating activity, of compounds of the invention.

Other properties and utilities of compounds of the invention can bedemonstrated using various in vitro and in vivo assays well-known tothose skilled in the art. For example, the in vivo potency of compoundsof the invention can be measured in an animal model such as theEinthoven model. Briefly, the bronchodilator activity of a compound isevaluated in an anesthetized animal (the Einthoven model), which usesventilation pressure as a surrogate measure of airway resistance. See,for example, Einthoven (1892) Pfugers Arch. 51:367-445; and Mohammed etal. (2000) Pulm Pharmacol Ther. 13(6):287-92, as well as Assay 3 whichdescribes a rat Einthoven model. In one embodiment, a compound of theinvention administered at a dose of 100 μg/ml in the rat Einthoven modelexhibits greater than or equal to 35% inhibition of thebronchoconstrictor response at 24 hours, and in another embodimentexhibits greater than or equal to 70% inhibition at 24 hours. Anotheruseful in vivo assay is the rat antisialagogue assay (for example, asdescribed in Assay 4).

The quaternary compounds of the invention also provide surprisingadvantages over the corresponding non-quaternary compounds, asmanifested, for example, in improved in vitro potency or improved invivo potency at 24 hours post-dosing. For example, both enantiomers ofthe non-quaternary compound:

exhibit a hM₃ K_(i) value of 1.5 and 2.9 nM (measured at 6 hours),respectively, while quaternary compounds of the invention, such as(S)-3-(carbamoyldiphenylmethyl)-1-methyl-1-(3-phenylsulfanylpropyl)pyrrolidinium(Example 2) and(R)-3-(carbamoyldiphenylmethyl)-1-methyl-1-(3-phenylsulfanylpropyl)pyrrolidinium(Example 5-11):

exhibit a hM₃ K_(i) value of less than 0.5 nM (measured at 6 hours).

Compounds of the invention are expected to be useful as therapeuticagents for treating medical conditions mediated by muscarinic receptors.Thus it is expected that patients suffering from a disease or disorderthat is treated by blocking the muscarinic receptor can be treated byadministering a therapeutically effective amount of a muscarinicreceptor antagonist of the invention. Such medical conditions include,by way of example, pulmonary disorders or diseases including thoseassociated with reversible airway obstruction, such as chronicobstructive pulmonary disease (e.g., chronic and wheezy bronchitis andemphysema), asthma, pulmonary fibrosis, allergic rhinitis, rhinorrhea,and the like. Other medical conditions that can be treated withmuscarinic receptor antagonists are genitourinary tract disorders, suchas overactive bladder or detrusor hyperactivity and their symptoms;gastrointestinal tract disorders, such as irritable bowel syndrome,diverticular disease, achalasia, gastrointestinal hypermotilitydisorders and diarrhea; cardiac arrhythmias, such as sinus bradycardia;Parkinson's disease; cognitive disorders, such as Alzheimer's disease;dismenorrhea; and the like.

The amount of active agent administered per dose or the total amountadministered per day may be predetermined or it may be determined on anindividual patient basis by taking into consideration numerous factors,including the nature and severity of the patient's condition, thecondition being treated, the age, weight, and general health of thepatient, the tolerance of the patient to the active agent, the route ofadministration, pharmacological considerations such as the activity,efficacy, pharmacokinetics and toxicology profiles of the active agentand any secondary agents being administered, and the like. Treatment ofa patient suffering from a disease or medical condition (such as COPD)can begin with a predetermined dosage or a dosage determined by thetreating physician, and will continue for a period of time necessary toprevent, ameliorate, suppress, or alleviate the symptoms of the diseaseor medical condition. Patients undergoing such treatment will typicallybe monitored on a routine basis to determine the effectiveness oftherapy. For example, in treating COPD, significant improvement inforced expiratory volume (measured in one second) may be used todetermine the effectiveness of treatment. Similar indicators for theother diseases and conditions described herein, are well-known to thoseskilled in the art, and are readily available to the treating physician.Continuous monitoring by the physician will insure that the optimalamount of active agent will be administered at any given time, as wellas facilitating the determination of the duration of treatment. This isof particular value when secondary agents are also being administered,as their selection, dosage, and duration of therapy may also requireadjustment. In this way, the treatment regimen and dosing schedule canbe adjusted over the course of therapy so that the lowest amount ofactive agent that exhibits the desired effectiveness is administeredand, further, that administration is continued only so long as isnecessary to successfully treat the disease or medical condition.

Accordingly, in one embodiment, compounds of the invention are usefulfor treating smooth muscle disorders in mammals, including humans andtheir companion animals (e.g., dogs, cats etc.). Such smooth muscledisorders include, by way of illustration, overactive bladder, chronicobstructive pulmonary disease and irritable bowel syndrome. Typically,suitable doses for treating smooth muscle disorders or other disordersmediated by muscarinic receptors will range from about 0.14 μg/kg/day toabout 7 mg/kg/day of active agent; including from about 0.15 μg/kg/dayto about 5 mg/kg/day. For an average 70 kg human, this would amount toabout 10 μg per day to about 500 mg per day of active agent.

In a specific embodiment, compounds of the invention are useful fortreating pulmonary or respiratory disorders, such as COPD or asthma, inmammals including humans, by administering to a patient atherapeutically effective amount of the compound. Generally, the dosefor treating a pulmonary disorder will range from about 10-1500 μg/day.The term “COPD” is understood by those of ordinary skill in the art toinclude a variety of respiratory conditions, including chronicobstructive bronchitis and emphysema, as exemplified by the teachings ofBarnes (2000) N. Engl. J. Med. 343:269-78, and references cited therein.When used to treat a pulmonary disorder, compounds of the invention areoptionally administered in combination with other therapeutic agentssuch as a β₂-adrenoreceptor agonist; a corticosteroid, a non-steroidalanti-inflammatory agent, or combinations thereof.

When administered by inhalation, compounds of the invention typicallyhave the effect of producing bronchodilation. Accordingly, in another ofits method aspects, the invention is directed to a method of producingbronchodilation in a patient, comprising administering to a patient abronchodilation-producing amount of a compound of the invention.Generally, the therapeutically effective dose for producingbronchodilation will range from about 10-1500 μg/day.

In another embodiment, compounds of the invention are used to treatoveractive bladder. When used to treat overactive bladder, a typicaldose will range from about 1.0-500 mg/day. In yet another embodiment,compounds of the invention are used to treat irritable bowel syndrome.When used to treat irritable bowel syndrome, compounds of the inventionwill typically be administered orally or rectally, and a typical dosewill range from about 1.0-500 mg/day.

Since compounds of this invention possess muscarinic receptor antagonistactivity, such compounds are also useful as research tools forinvestigating or studying biological systems or samples havingmuscarinic receptors. Any suitable biological system or sample havingM₁, M₂, M₃, M₄ and/or M₅ muscarinic receptors may be employed in suchstudies which may be conducted either in vitro or in vivo.Representative biological systems or samples suitable for such studiesinclude, but are not limited to, cells, cellular extracts, plasmamembranes, tissue samples, isolated organs, mammals (such as mice, rats,guinea pigs, rabbits, dogs, pigs, humans, and so forth), and the like,with mammals being of particular interest. In one particular embodimentof the invention a muscarinic receptor in a mammal is antagonized byadministering a muscarinic receptor-antagonizing amount of a compound ofthe invention. Compounds of the invention can also be used as researchtools by conducting biological assays using such compounds.

When used as a research tool, a biological system or sample comprising amuscarinic receptor is typically contacted with a muscarinicreceptor-antagonizing amount of a compound of the invention. After thebiological system or sample is exposed to the compound, the effects ofantagonizing the muscarinic receptor are determined using conventionalprocedures and equipment, such as by measuring binding in a radioligandbinding assays or ligand-mediated changes in a functional assay or bydetermining the amount of bronchoprotection provided by the compound ina bronchoprotection assay in a mammal. Exposure encompasses contactingcells or tissue with the compound, administering the compound to amammal, for example by i.p. or i.v. administration, and so forth. Thisdetermining step may comprise measuring a response, i.e., a quantitativeanalysis or may comprise an observation, i.e., a qualitative analysis.Measuring a response involves, for example, determining the effects ofthe compound on the biological system or sample using conventionalprocedures and equipment, such as radioligand binding assays andmeasuring ligand-mediated changes in functional assays. The assayresults can be used to determine the activity level as well as theamount of compound necessary to achieve the desired result, i.e., amuscarinic-antagonizing amount. Typically, the determining step willinvolve determining the muscarinic receptor ligand-mediated effects.

Additionally, compounds of the invention can be used as research toolsfor evaluating other chemical compounds, and thus are also useful inscreening assays to discover, for example, new compounds havingmuscarinic receptor binding activity. In this manner, a compound of theinvention is used as a standard in an assay to allow comparison of theresults obtained with a test compound and with compounds of theinvention to identify those test compounds that have about equal orsuperior binding, if any. For example, muscarinic receptor binding data(as determined, for example, by in vitro radioligand displacementassays) for a test compound or a group of test compounds is compared tothe muscarinic receptor binding data for a compound of the invention toidentify those test compounds that have the desired properties, e.g.,test compounds having binding about equal or superior to a compound ofthe invention, if any. Alternatively, for example, bronchoprotectiveeffects can be determined for test compounds and a compound of theinvention in a bronchoprotection assay in a mammal and this datacompared to identify test compounds providing about equal or superiorbronchoprotective effects. This aspect of the invention includes, asseparate embodiments, both the generation of comparison data (using theappropriate assays) and the analysis of the test data to identify testcompounds of interest. Thus, a test compound can be evaluating in abiological assay, by a method comprising the steps of: (a) conducting abiological assay with a test compound to provide a first assay value;(b) conducting the biological assay with a compound of the invention toprovide a second assay value; wherein step (a) is conducted eitherbefore, after or concurrently with step (b); and (c) comparing the firstassay value from step (a) with the second assay value from step (b).Exemplary biological assays include muscarinic receptor binding assays.

EXAMPLES

The following Preparations and Examples are provided to illustratespecific embodiments of the invention. These specific embodiments,however, are not intended to limit the scope of the invention in any wayunless specifically indicated.

The following abbreviations have the following meanings unless otherwiseindicated and any other abbreviations used herein and not defined havetheir standard meaning:

AC adenylyl cyclase

ACN acetonitrile

BSA bovine serum albumin

cAMP 3′-5′ cyclic adenosine monophosphate

CHO Chinese hamster ovary

cM₅ cloned chimpanzee M₅ receptor

DCM dichloromethane (i.e., methylene chloride)

DIPEA N,N-diisopropylethylamine

dPBS Dulbecco's phosphate buffered saline

DMA N,N-dimethylacetamide

EDTA ethylenediamine tetraacetic acid

EtOAc ethyl acetate

EtOH ethanol

FBS fetal bovine serum

FLIPR fluorometric imaging plate reader

HBSS Hank's Buffered Salt Solution

HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid

hM₁ cloned human M₁ receptor

hM₂ cloned human M₂ receptor

hM₃ cloned human M₃ receptor

hM₄ cloned human M₄ receptor

hM₅ cloned human M₅ receptor

IPAc isopropyl acetate

MCh methylcholine

MeOH methanol

MTBE methyl t-butyl ether

TFA trifluoroacetic acid

TFA salt trifluoroacetate salt

THF tetrahydrofuran

Any other abbreviations used herein but not defined have their standard,generally accepted meaning. Unless noted otherwise, all materials, suchas reagents, starting materials and solvents, were purchased fromcommercial suppliers (such as Sigma-Aldrich, Fluka Riedel-de Haen, andthe like) and were used without further purification. Reactions were rununder nitrogen atmosphere, unless noted otherwise. Progress of reactionmixtures was monitored by thin layer chromatography (TLC), analyticalhigh performance liquid chromatography (anal. HPLC), and massspectrometry, the details of which are given below and separately inspecific examples of reactions. Reaction mixtures were worked up asdescribed specifically in each reaction; commonly they were purified byextraction and other purification methods such as temperature-, andsolvent-dependent crystallization, and precipitation. In addition,reaction mixtures were routinely purified by preparative HPLC.

Preparation 1 2,2-Diphenyl-2-(S)-pyrrolidin-3-ylacetamide

(S)-1-Benzyl-3-(p-toluenesulfonyloxy)pyrrolidine (1a): To a stirredsolution of (S)-1-benzyl-3-pyrrolidinol (44.3 g, 0.25 mol) and1,4-diazabicyclo[2.2.2]octane (33.7 g, 0.3 mol) in 250 mL of MTBE undernitrogen at 0° C., was added p-toluenesulfonyl chloride (52.4 g, 0.275mol) portion-wise over 20 minutes. The mixture was stirred at 0° C. for1 hour. The ice bath was removed and the mixture was stirred at ambienttemperature overnight (20±5 hours). EtOAc (100 mL) was added, followedby saturated aqueous sodium bicarbonate solution (250 mL). The resultingmixture was stirred at ambient temperature for 1 hour. The layers wereseparated and the organic layer was washed with saturated aqueous sodiumbicarbonate solution (250 mL); saturated aqueous NH₄Cl solution (250mL); saturated aqueous NaCl solution (250 mL); and then dried oversodium sulfate (80 g). The sodium sulfate was filtered off and washedwith EtOAc (20 mL) and the solvent was removed in vacuo to give 78.2 gof intermediate (1a) as an off-white solid (94% yield). HPLC analysiswas conducted using a YMC ODSA C18 4.6×50 mm column, having a 5.0 micronparticle size. Detection was by UV absorbance at 220 nm. The mobilephases employed were as follows (percentage by volume): A:MeOH/water/TFA (10/90/0.1; and B: MeOH/water/TFA (90/10/0.1). Using aflow rate of 4.0 mL/min of 0 to 100% B in A over 5 minutes, thisintermediate was determined to have a purity of 95%.

(S)-1-Benzyl-3-(1-cyano-1,1-diphenylmethyl)-pyrrolidine (1b): To astirred solution of diphenylacetonitrile (12.18 g, 61.8 mmol) inanhydrous THF (120 mL) at 0° C., potassium tert-butoxide (10.60 g, 94.6mmol) was added over 5 min. The mixture was stirred at 0° C. for 1 hour.To the mixture at 0° C. was added intermediate (1a) (20.48 g, 61.3 mmol)in one portion. The cold bath was removed and the mixture was stirredfor 5-10 minutes at which time the mixture had become a brownhomogeneous solution. The mixture was then heated at 40° C. overnight(20±5 hours). The mixture (bright yellow suspension) was allowed to coolto ambient temperature before adding water (150 mL). Most of the THF wasthen removed in vacuo and IPAc (200 mL) was added. The layers wereseparated and the organic layer was washed with saturated aqueous NH₄Clsolution (150 mL); saturated aqueous NaCl solution (150 mL); and thendried over sodium sulfate (50 g). The sodium sulfate was filtered offand washed with IPAc (20 mL) and the solvent was removed in vacuo togive 23.88 g of intermediate (1b) as a light brown oil (>99% yield).Intermediate (1b) was determined to have a purity of 75% (contaminatedmainly with excess diphenylacetonitrile) using the HPLC method describedabove.

(S)-3-(1-Cyano-1,1-diphenylmethyl)pyrrolidine (Ic): Intermediate (Ib)was dissolved in IPAc (approximately 1 g/10 mL) and the solution wasmixed with an equal volume of 1N aqueous HCl. The resulting layers wereseparated and the aqueous layer was extracted with an equal volume ofIPAc. The organic layers were combined, dried over sodium sulfate andfiltered. The solvent was removed in vacuo to afford(S)-1-benzyl-3-(1-cyano-1,1-diphenylmethyl)pyrrolidine hydrochloride asa light yellow foamy solid. To a stirred solution of(S)-1-benzyl-3-(1-cyano-1,1-diphenylmethyl)pyrrolidine hydrochloride(8.55 g, 21.98 mmol) in MeOH (44 mL) was added palladium on carbon (1.71g) and ammonium formate (6.93 g, 109.9 mmol). The mixture was heated to50° C. with stirring for 3 hours. The mixture was cooled to ambienttemperature and water (20 mL) was added. The resulting mixture wasfiltered through a pad of Celite®, washing with MeOH (20 mL). Thefiltrate was collected and most of the MeOH was removed in vacuo. Theresidue was mixed with IPAc (100 mL) and 10% aqueous sodium carbonate(50 mL). The resulting layers were separated and the aqueous layer wasextracted with IPAc (50 mL). The organic layers were combined and driedover sodium sulfate (20 g). The sodium sulfate was filtered off andwashed with IPAc (20 mL). The solvent was removed in vacuo to afford5.75 g of intermediate (1c) as a light yellow oil (99.7% yield, 71%purity by HPLC).

2,2-Diphenyl-2-(S)-pyrrolidin-3-ylacetamide: A 200 mL flask with amagnetic stir bar and a nitrogen inlet was charged with intermediate(1c) (2.51 g) and 80% H₂SO₄ (19.2 mL; pre-prepared with 16 mL of 96%H₂SO₄ and 3.2 mL of H₂O). The mixture was then heated at 90° C. for 24hours or until the starting material was consumed as indicated by HPLC.The mixture was allowed to cool to ambient temperature and then pouredonto ice (approximately 50 mL by volume). A 50% aqueous NaOH solutionwas added slowly to the mixture with stirring over an ice bath until thepH was about 12. DCM (200 mL) was added and mixed with the aqueoussolution at which time sodium sulfate precipitated out and was filteredoff. The filtrate was collected and the layers were separated. Theaqueous layer was extracted with DCM (100 mL) and the organic layerswere combined and dried with over sodium sulfate (5 g). The sodiumsulfate was filtered off and washed with DCM (10 mL). The solvent wasremoved in vacuo to give the crude product as a light yellow foamy solid(ca. 2.2 g, 86% purity by HPLC).

The crude product was dissolved in EtOH (18 mL) with stirring. To thissolution was added a warm solution of L-tartaric acid (1.8 g) in EtOH(14 mL) and the resulting mixture was stirred overnight (15±5 hours).The resulting precipitate was isolated by filtration to give anoff-white solid (ca. 3.2 g, >95% purity by HPLC). MeOH (15 mL) was addedto this solid and the resulting slurry was stirred at 70° C. overnight(15 hours). The slurry was allowed to cool to ambient temperature and awhite solid (˜2.6 g, >99% purity by HPLC) was obtained after filtration.To this solid was added EtOAc (30 mL) and 1 N aqueous NaOH (25 mL). Thismixture was mixed until two distinct layers formed and then the layerswere separated and the aqueous layer was extracted with EtOAc (20 mL).The organic layers were combined and dried over sodium sulfate (10 g).The sodium sulfate was removed by filtration and the solvent wasevaporated in vacuo to afford 1.55 g of the title compound as anoff-white foamy solid (58% yield).

HPLC analysis was conducted using an Inertsil OCD-2 C18 column.Detection was by UV absorbance at 254 nm. The mobile phases employedwere as follows (percentage by volume): A: MeOH/water/TFA (5/95/0.1);and B: MeOH/water/TFA (95/5/0.1). Using a flow rate of 1.0 mL/min of 0to 100% B in A over 15 minutes, this compound was determined to have apurity of >99%.

Preparation 22-[(S)-1-(3-Phenoxypropyl)pyrrolidin-3-yl]-2,2-diphenylacetamide

Into a vial was added 2,2-diphenyl-2-(S)-pyrrolidin-3-ylacetamide (42.0mg, 0.15 mmol; prepared as described in Preparation 1) and3-bromopropoxybenzene (23.7 μL, 0.15 mmol) in ACN (1 mL) and DIPEA (78μL, 0.45 mmol). The mixture was heated to 70° C. overnight. Afterovernight, the reaction was cooled to room temperature and concentrated.The mixture was chromatographed on reverse-phase HPLC (gradient elution,10-50% ACN/H₂O) to afford 49.0 mg (92% purity) of the title compound(56% yield) as a trifluoroacetate salt. MS m/z: [M+H⁺] calcd forC₂₇H₃₀N₂O₂, 415.23. found 415.2.

Example 1(S)-3-(Carbamoyldiphenylmethyl)-1-methyl-1-(3-phenoxypropyl)pyrrolidinium

Into a vial was added2-[(S)-1-(3-phenoxypropyl)pyrrolidin-3-yl]-2,2-diphenylacetamide (79 mg,0.15 mmol; prepared as described in Preparation 2) in DMA (1 mL) andmethyl iodide (0.093 mL, 1.5 mmol) and the mixture was stirred for 2hours at 80° C. The mixture was then cooled to room temperature andconcentrated. The mixture was chromatographed on reverse-phase HPLC(gradient elution, 10-50% ACN/H₂O) to afford 35.0 mg (85% purity) of thetitle compound (36% yield) as a TFA salt. MS m/z: [M⁺] calcd forC₂₈H₃₃N₂O₂, 429.25. found 429.6. ¹H NMR (CDCl₃, 300 MHz) δ (ppm): 2.29(2H, m), 2.68 (1H, s), 2.90 (5H, s), 2.98 (5H, s), 3.37 (2H, m), 3.59(2H, m) 3.95 (H, m), 4.06 (2H, t), 5.93 (2H, s), 6.9 (4H, m), 7.28 (7H,m).

Preparation 3 2-((S)-1-Methylpyrrolidin-3-yl)-2,2-diphenylacetamide

To a round-bottom flask was added2,2-diphenyl-2-(S)-pyrrolidin-3-ylacetamide (2.8 g, 10.0 mmol; preparedas described in Preparation 1), MeOH (30 mL) and then formaldehyde (2mL, 30% in water, 24.6 mmol) with stirring. The solution was then purgedwith nitrogen and then with hydrogen. The mixture was stirred overnightunder hydrogen. The mixture was then filtered through a pad of Celite®,concentrated in vacuo, then taken up in H₂O/ACN (1:1 mixture, 1 gsolid/20 mL solvent mixture). The mixture was stirred overnight at roomtemperature. Finally the mixture was filtered and then allowed to dry toafford 2.7 g of the title compound as an off-white foamy solid (96.4%yield). MS m/z: [M+H⁺] calcd for C₁₉H₂₂N₂O, 294.39. found 295.0. ¹H NMR(CDCl₃, 300 MHz) δ (ppm): 1.73 (1H, s), 1.97 (2H, m), 2.27 (3H, s), 2.42(1H, q), 2.55 (1H, m), 2.67 (1H, t), 2.75 (1H, m), 3.46 (1H, m), 5.63(1H, br s), 7.26 (8H, m), 7.41 (2H, m).

Example 2(S)-3-(Carbamoyldiphenylmethyl)-1-methyl-1-(3-phenylsulfanylpropyl)pyrrolidinium

Into a vial was added2-((S)-1-methylpyrrolidin-3-yl)-2,2-diphenylacetamide (300 mg, 1.0 mmol;prepared as described in Preparation 3) in DMA (10 mL) and3-chloropropylsulfanylbenzene (200 mg, 1.0 mmol). Sodium iodide (30 mg,0.2 mmol) was added to the mixture. The mixture was degassed undernitrogen and then was heated to 90° C. overnight. The mixture was thenconcentrated and chromatographed on reverse-phase HPLC (gradientelution, 10-50% ACN/H₂O) to afford 49.0 mg (92% purity) of the titlecompound as a TFA salt. MS m/z: [M⁺] calcd for C₂₈H₃₃N₂OS: 445.23.found: 445.1. ¹H NMR (CDCl₃, 300 MHz) δ (ppm): 1.97 (1H, m), 2.20 (1H,m), 2.70 (2H, s), 2.88 (2H, s), 2.93 (2H, s), 3.3 (1H, m), 3.4 (1H, m),3.52 (1H, m), 3.89 (2H, m), 4.03 (2H, m), 4.30 (1H, t), 5.63 (1H, d)5.94 (1H, s) 7.20 (2H, m), 7.35 (13H, m).

Preparation 4(S)-1-(3-Bromopropyl)-3-(carbamoyldiphenylmethyl)-1-methylpyrrolidiniumBromide

To a round-bottom flask was dissolved2,2-diphenyl-2-(S)-pyrrolidin-3-ylacetamide (2.8 g, 10 mmol; prepared asdescribed in Preparation 1) and 1,3-dibromopropane (6.8 mL, 50 mmol) inDMA (30 mL). The mixture was stirred and placed in an oil bath at 80° C.for 2 hours. The mixture was cooled to room temperature and ethyl ether(200 mL) was added with stirring. Immediate formation of whiteprecipitate occurred. The mixture was stirred overnight. The whiteprecipitate was filtered and dried in a vacuum dessicator to afford 6.6g of the title compound as a white solid (Yield=95%). MS m/z: [M⁺] calcdfor C₂₂H₂₈BrN₂O₂, 415.14. found 417.2. ¹H NMR (CDCl₃, 300 MHz) δ (ppm):1.86 (3H, s), 2.20 (2H, m), 2.40 (1H, m), 2.73 (1H, m), 2.78 (1H, s),3.49 (6H, m), 3.89 (1H, m), 4.06 (2H, m), 4.38 (1H, t), 5.80 (1H, br d),6.32 (1H, br s), 7.34 (10H, m).

Example 3 (S)-3-(Carbamoyldiphenylmethyl)-1-[3-(3,5-difluorophenoxy)propyl]-1-methyl-pyrrolidinium

To a round-bottom flask was added(S)-1-(3-bromopropyl)-3-(carbamoyldiphenylmethyl)-1-methylpyrrolidiniumbromide (900 mg, 2.2 mmol; prepared as described in Preparation 4) inDMA (10 mL). To the mixture was added 3,5-difluorophenol (288 mg, 2.2mmol), followed by potassium carbonate (0.5 g, 3.6 mmol), which wasstirred vigorously. The mixture was stirred overnight at roomtemperature. The mixture was then concentrated and chromatographed onreverse-phase HPLC (gradient elution, 10-50% ACN/H₂O) to afford 75.0 mg(99% purity) of the title compound (6% yield) as a TFA salt. MS m/z:[M⁺] calcd for C₂₈H₃₁F₂N₂O₂: 465.24. found: 465.2. ¹H NMR (CDCl₃, 300MHz) δ (ppm): 2.10 (1H, s), 2.19 (2H, s), 2.67 (3H, s), 2.80 (1H, s),3.25 (3H, s), 3.53 (1H, t), 3.67 (2H, s), 3.87 (2H, s), 4.01 (2H, s),4.20 (1H, t), 5.80 (1H, d), 6.38 (3H, m), 6.53 (1H, d).

Preparation 5 1-(3-Bromopropylsulfanyl)-3,5-difluorobenzene

To a round-bottom flask was added 3,5-difluorobenzenethiol (4.9 g, 33.8mmol) in ACN (50 mL). 3-Bromo-1-propanol (4.7 g, 34.0 mmol) andpotassium carbonate (6.4 g, 46 mmol) were added, and the mixture wasstirred at room temperature for 3 hours. The mixture was then filteredthrough a pad of Celite® and the solvent was removed under reducedpressure. The crude material was then dissolved in anhydrous DCM (100mL). Triphenylphosphine dibromide (25 g, 59 mmol) was added to themixture, which was then stirred for 2 hours. The solvent was removedunder reduced pressure. The residue was suspended in EtOAc (200 mL),stirred for 1 hour, and filtered to remove solids. The filtrate wasconcentrated under reduced pressure, then diluted in 50% EtOAc/hexanes(200 mL), and filtered through a pad of Celite® and silica gel. Thesolution was then concentrated under reduced pressure to afford thetitle compound (5.8 g).

Example 4(1R,3S)-3-(Carbamoyldiphenylmethyl)-1-[3-(3,5-difluorophenylsulfanyl)propyl]-1-methylpyrrolidinium

2,2-Diphenyl-2-(S)-pyrrolidin-3-ylacetamide (3.5 g, 12.5 mmol; preparedas described in Preparation 1) in DMA (33 mL) was combined with DIPEA(3.5 mL, 20 mmol), followed by the addition of1-(3-bromopropylsulfanyl)-3,5-difluorobenzene (3.5 g, 13.1 mmol;prepared as described in Preparation 5). The mixture was stirred for 14hours at room temperature. Methyl iodide (3.1 mL, 50 mmol) was added andthe mixture was stirred at 70° C. for 2 hours. The mixture was thenconcentrated in vacuo. The resulting product was a mixture of 2diastereomers at the quaternary amine center. The diastereomers wereseparated by reverse phase HPLC on a LUNA C-18 Column using a 37%(ACN/H₂O/0.1% TFA) isocratic gradient over 45 minutes, to afford 1.5 gof the title compound as a TFA salt. MS m/z: [M⁺] calcd forC₂₈H₃₁F₂N₂OS, 481.21. found 481.2.

Example 5

Following the procedures described in the previous examples, andsubstituting the appropriate starting materials and reagents, compounds5-1 to 5-23, having the following formula were also prepared as TFAsalts:

Ex. Q 5-1  —(CH₂)₂— 5-2  —(CH₂)₃— 5-3  —(CH₂)₆— 5-4  —(CH₂)₂—C(O)O— 5-5 —(CH₂)₂—O—CH₂— 5-6  —(CH₂)₂—NHC(O)— 5-7  —(CH₂)₄— 5-8  —(CH₂)₂—OC(O)—5-9  —(CH₂)₄— 5-10 —(CH₂)₃—O— 5-11 —(CH₂)₃—S— 5-12 —(CH₂)₂—OC(O)— 5-13—(CH₂)₃—C(O)— 5-14 —(CH₂)₅— 5-15 —(CH₂)₂—NHC(O)NH— 5-16 —CH₂—C(O)NH—CH₂—5-17 —CH₂—C(O)O—CH₂— 5-18 —(CH₂)₃—C(N—O—CH₃)— 5-19—(CH₂)₃—C(N—O-benzyl)— 5-20 —(CH₂)₃—S— 5-21 —(CH₂)₃—S— 5-22 —(CH₂)₂—C≡C—5-23 —(CH₂)₂—CH═CH— 5-24 —CH₂—C═N—O— 5-25 —(CH₂)₂—S—S—(5-1) 3-(carbamoyldiphenylmethyl)-1-methyl-1-phenethylpyrrolidinium. MSm/z: [M⁺] calcd for C₂₇H₃₁N₂O, 399.24. found 399.0.(5-2)3-(carbamoyldiphenylmethyl)-1-methyl-1-(3-phenylpropyl)pyrrolidinium. MSm/z: [M⁺] calcd for C₂₈H₃₃N₂O, 413.26. found 413.0.(5-3)3-(carbamoyldiphenylmethyl)-1-methyl-1-(6-phenylhexyl)pyrrolidinium. MSm/z: [M⁺] calcd for C₃₁H₃₉N₂O, 455.31. found 455.4.(5-4)3-[(S)-3-(carbamoyldiphenylmethyl)-1-methylpyrrolidin-1-yl]-propionicacid phenyl ester. MS m/z: [M⁺] calcd for C₂₈H₃₁N₂O₃, 443.23. found443.2.(5-5)2-[(S)-1-(2-benzyloxyethyl)-1-methylpyrrolidin-3-yl]-2,2-diphenylacetamide.MS m/z: [M⁺] calcd for C₂₈H₃₃N₂O₂, 429.25. found 429.2.(5-6)N-{2-[(S)-3-(carbamoyldiphenylmethyl)-1-methylpyrrolidin-1-yl]ethyl}benzamide.MS m/z: [M⁺] calcd for C₂₈H₃₂N₃O₂, 442.25. found 442.2.(5-7)2-[(S)-1-methyl-1-(4-phenylbutyl)pyrrolidin-3-yl]-2,2-diphenylacetamide.MS m/z: [M⁺] calcd for C₂₉H₃₅N₂O, 427.28. found 427.2.(5-8) benzoic acid2-[(S)-3-(carbamoyldiphenylmethyl)-1-methylpyrrolidin-1-yl]ethyl ester.MS m/z: [M⁺] calcd for C₂₈H₃₁N₂O₃, 443.23. found 443.2.(5-9)(R)-3-(carbamoyldiphenylmethyl)-1-methyl-1-(4-phenylbutyl)pyrrolidinium.MS m/z: [M⁺] calcd for C₂₉H₃₅N₂O, 427.28. found 427.2.(5-10)(R)-3-(carbamoyldiphenylmethyl)-1-methyl-1-(3-phenoxypropyl)pyrrolidinium.MS m/z: [M⁺] calcd for C₂₈H₃₃N₂O₂, 429.25. found 429.2.(5-11)(R)-3-(carbamoyldiphenylmethyl)-1-methyl-1-(3-phenylsulfanylpropyl)pyrrolidinium.MS m/z: [M⁺] calcd for C₂₈H₃₃N₂OS, 445.23. found 445.2.(5-12)(R)-1-(2-benzoyloxyethyl)-3-(carbamoyldiphenylmethyl)-1-methylpyrrolidinium.MS m/z: [M⁺] calcd for C₂₈H₃₁N₂O₃, 443.23. found 443.2.(5-13)(S)-3-(carbamoyldiphenylmethyl)-1-methyl-1-(4-oxo-4-phenylbutyl)pyrrolidinium.MS m/z: [M⁺] calcd for C₂₉H₃₃N₂O₂, 441.25. found 441.2.(5-14)(S)-3-(carbamoyldiphenylmethyl)-1-methyl-1-(5-phenylpentyl)pyrrolidinium.MS m/z: [M⁺] calcd for C₃₀H₃₇N₂O, 441.29. found 441.2.(5-15)(S)-3-(carbamoyldiphenylmethyl)-1-methyl-1-[2-(3-phenylureido)ethyl]pyrrolidinium.MS m/z: [M⁺] calcd for C₂₈H₃₃N₄O₂, 457.26. found 457.2.(5-16)(S)-1-(benzylcarbamoylmethyl)-3-(carbamoyldiphenylmethyl)-1-methylpyrrolidinium.MS m/z: [M⁺] calcd for C₂₈H₃₂N₃O₂, 442.25. found 442.2.(5-17)(S)-1-benzyloxycarbonylmethyl-3-(carbamoyldiphenylmethyl)-1-methylpyrrolidinium.MS m/z: [M⁺] calcd for C₂₈H₃₁N₂O₃, 443.23. found 443.2.(5-18)2-((S)-1-{4-[(E)-methoxyimino]-4-phenylbutyl}-1-methylpyrrolidin-3-yl)-2,2-diphenylacetamide.MS m/z: [M⁺] calcd for C₃₀H₃₆N₃O₂, 470.28. found 470.2.(5-19)2-((S)-1-{4-[(E)-benzyloxyimino]-4-phenylbutyl}-1-methylpyrrolidin-3-yl)-2,2-diphenylacetamide.MS m/z: [M⁺] calcd for C₃₆H₄₀N₃O₂, 546.31. found 546.4.(5-20)2-[(1S,3S)-1-methyl-1-(3-phenylsulfanylpropyl)-pyrrolidin-3-yl]-2,2-diphenylacetamide.MS m/z: [M⁺] calcd for C₂₈H₃₃N₂OS, 445.23. found 445.2.(5-21)2-[(1R,3S)-1-methyl-1-(3-phenylsulfanylpropyl)-pyrrolidin-3-yl]-2,2-diphenylacetamide.MS m/z: [M⁺] calcd for C₂₈H₃₃N₂OS, 445.23. found 445.2.(5-22)(S)-3-(carbamoyldiphenylmethyl)-1-methyl-1-(4-phenyl-but-3-ynyl)pyrrolidinium.MS m/z: [M⁺] calcd for C₂₉H₃₁N₂O, 423.24. found 423.2.(5-23)(S)-3-(carbamoyldiphenylmethyl)-1-methyl-1-((E)-4-phenyl-but-3-enyl)pyrrolidinium.MS m/z: [M⁺] calcd for C₂₉H₃₃N₂O, 425.26. found 425.2.(5-24)(S)-3-(carbamoyldiphenylmethyl)-1-methyl-1-(2-phenoxyiminoethyl)pyrrolidinium.MS m/z: [M⁺] calcd for C₂₇H₃₀N₃O₂, 429.23. found 428.2.(5-25)(S)-3-(carbamoyldiphenylmethyl)-1-methyl-1-(3-phenyldisulfanylpropyl)pyrrolidinium MS m/z: [M⁺] calcd for C₂₈H₃₃N2OS₂, 478.20. found 477.2.

Example 6

Following the procedures described in the previous examples, andsubstituting the appropriate starting materials and reagents, compounds6-1 to 6-30, having the following formula were also prepared as TFAsalts:

Ex. Q R⁶ 6-1  —(CH₂)₂— —N⁺(O)O 6-2  —(CH₂)₃—O— —F 6-3  —(CH₂)₂— —OH 6-4 —(CH₂)₂—OC(O)— —OCF₃ 6-5  —(CH₂)₂—NHC(O)— —OCH₃ 6-6  —(CH₂)₂—OC(O)——N(CH₃)₂ 6-7  —(CH₂)₂—OC(O)— —OCH₃ 6-8  —(CH₂)₄— —OCH₃ 6-9  —(CH₂)₃—O——CH₃ 6-10 —(CH₂)₃—O— —C(O)OCH₃ 6-11 —CH₂—CH(OH)—CH₂—O— F 6-12 —(CH₂)₃—O——OH 6-13 —(CH₂)₃—O— —Cl 6-14 —(CH₂)₃—S— —Br 6-15 —(CH₂)₃—S— —Cl 6-16—(CH₂)₃—S— —OCH₃ 6-17 —(CH₂)₃—C(O)— —Br 6-18 —(CH₂)₃—O— —N⁺(O)O 6-19—(CH₂)₃—O— —OCH₃ 6-20 —(CH₂)₃—C(O)— —F 6-21 —(CH₂)₃—C(O)— —OH 6-22—(CH₂)₃—C(O)— —OCH₃ 6-23 —(CH₂)₃—C(O)— —CH₃ 6-24 —(CH₂)₃—S— —NHC(O)CH₃6-25 —(CH₂)₃—S— —S—CH₃ 6-26 —(CH₂)₃—S— —F 6-27 —(CH₂)₃—S— —CH₃ 6-28—(CH₂)₃—O— —S—CH₃ 6-29 —(CH₂)₃—C(O)— —Cl 6-30 —(CH₂)₃—S— —OH(6-1)3-(carbamoyldiphenylmethyl)-1-methyl-1-[2-(4-nitrophenyl)ethyl]pyrrolidinium.MS m/z: [M⁺] calcd for C₂₇H₃₀N₃O₃, 444.23. found 444.0.(6-2)3-(carbamoyldiphenylmethyl)-1-[3-(4-fluorophenoxy)propyl]-1-methylpyrrolidinium.MS m/z: [M⁺] calcd for C₂₈H₃₂FN₂O₂, 447.24. found 447.0. ¹H NMR (CDCl₃,300 MHz) δ (ppm): 2.20 (3H, m), 2.73 (4H, d), 2.90 (1H, m), 3.35 (5H,s), 3.53 (1H, t), 3.68 (2H, t) 4.29 (1H, t), 4.31 (1H, t), 5.74 (1H, s),6.15 (1H, d), 6.77 (2H, m) 6.79 (2H, m) 7.26 (11H, m).(6-3)3-(carbamoyldiphenylmethyl)-1-[2-(4-hydroxyphenyl)ethyl]-1-methylpyrrolidinium.MS m/z: [M⁺] calcd for C₂₇H₃₁N₂O₂, 415.24. found 415.0.(6-4) 4-trifluoromethoxybenzoic acid2-[(S)-3-(carbamoyldiphenylmethyl)-1-methylpyrrolidin-1-yl]ethyl ester.MS m/z: [M⁺] calcd for C₂₉H₃₀F₃N₂O₄, 527.22. found 527.2.(6-5)N-{2-[(S)-3-(carbamoyldiphenylmethyl)-1-methylpyrrolidin-1-yl]ethyl}-4-methoxybenzamide.MS m/z: [M⁺] calcd for C₂₉H₃₄N₃O₃, 472.26. found 472.2.(6-6) 4-dimethylaminobenzoic acid2-[(S)-3-(carbamoyldiphenylmethyl)-1-methylpyrrolidin-1-yl]ethyl ester.MS m/z: [M⁺] calcd for C₃₀H₃₆N₃O₃, 486.28. found 486.2.(6-7) 4-methoxybenzoic acid2-[(S)-3-(carbamoyldiphenylmethyl)-1-methylpyrrolidin-1-yl]ethyl ester.MS m/z: [M⁺] calcd for C₂₉H₃₃N₂O₄, 473.24. found 473.2.(6-8)2-{(S)-1-[4-(4-methoxyphenyl)butyl]-1-methylpyrrolidin-3-yl}-2,2-diphenylacetamide.MS m/z: [M⁺] calcd for C₃₀H₃₇N₂O₂, 457.29. found 457.4.(6-9)2-[(S)-1-methyl-1-(3-p-tolyloxypropyl)pyrrolidin-3-yl]-2,2-diphenylacetamide.MS m/z: [M⁺] calcd for C₂₉H₃₅N₂O₂, 443.27. found 443.2.(6-10)4-{3-[(S)-3-(carbamoyldiphenylmethyl)-1-methylpyrrolidin-1-yl]propoxy}benzoicacid methyl ester. MS m/z: [M⁺] calcd for C₃₀H₃₅N₂O₄, 487.26. found487.2.(6-11)2-{(S)-1-[(S)-3-(4-fluorophenoxy)-2-hydroxypropyl]-1-methylpyrrolidin-3-yl}-2,2-diphenylacetamide.MS m/z: [M⁺] calcd for C₂₈H₃₂FN₂O₃, 463.24. found 463.2.(6-12)2-{(S)-1-[3-(4-hydroxyphenoxy)propyl]-1-methylpyrrolidin-3-yl}-2,2-diphenylacetamide.MS m/z: [M⁺] calcd for C₂₈H₃₃N₂O₃, 445.25. found 445.2.(6-13)2-{(S)-1-[3-(4-chlorophenoxy)propyl]-1-methylpyrrolidin-3-yl}-2,2-diphenylacetamide.MS m/z: [M⁺] calcd for C₂₈H₃₂ClN₂O₂, 463.22. found 463.2.(6-14)2-{(S)-1-[3-(4-bromophenylsulfanyl)propyl]-1-methylpyrrolidin-3-yl}-2,2-diphenylacetamide.MS m/z: [M⁺] calcd for C₂₈H₃₂BrN₂OS, 523.14. found 523.2.(6-15)2-{(S)-1-[3-(4-chlorophenylsulfanyl)propyl]-1-methylpyrrolidin-3-yl}-2,2-diphenylacetamide.MS m/z: [M⁺] calcd for C₂₈H₃₂ClN₂OS, 479.19. found 479.2.(6-16)2-{(S)-1-[3-(4-methoxyphenylsulfanyl)propyl]-1-methylpyrrolidin-3-yl}-2,2-diphenylacetamide.MS m/z: [M⁺] calcd for C₂₉H₃₅N₂O₂S, 475.24. found 475.2.(6-17)2-{1-[4-(4-bromophenyl)-4-oxo-butyl]-1-methylpyrrolidin-3-yl}-2,2-diphenylacetamide.MS m/z: [M⁺] calcd for C₂₉H₃₂BrN₂O₂, 519.16. found 519.2.(6-18)2-{1-methyl-1-[3-(4-nitrophenoxy)propyl]pyrrolidin-3-yl}-2,2-diphenylacetamide.MS m/z: [M⁺] calcd for C₂₈H₃₂N₃O₄, 474.24. found 474.2.(6-19)2-{1-[3-(4-methoxyphenoxy)propyl]-1-methylpyrrolidin-3-yl}-2,2-diphenylacetamide.MS m/z: [M⁺] calcd for C₂₉H₃₅N₂O₃, 459.26. found 459.2.(6-20)(S)-3-(carbamoyldiphenylmethyl)-1-[4-(4-fluorophenyl)-4-oxobutyl]-1-methylpyrrolidinium.MS m/z: [M⁺] calcd for C₂₉H₃₂FN₂O₂, 459.24. found 459.2.(6-21)(S)-3-(carbamoyldiphenylmethyl)-1-[4-(4-hydroxyphenyl)-4-oxobutyl]-1-methylpyrrolidinium.MS m/z: [M⁺] calcd for C₂₉H₃₃N₂O₃, 457.25. found 457.2.(6-22)(S)-3-(carbamoyldiphenylmethyl)-1-[4-(4-methoxyphenyl)-4-oxobutyl]-1-methylpyrrolidinium.MS m/z: [M⁺] calcd for C₃₀H₃₅N₂O₃, 471.26. found 471.2.(6-23)(S)-3-(carbamoyldiphenylmethyl)-1-methyl-1-(4-oxo-4-p-tolylbutyl)pyrrolidinium.MS m/z: [M⁺] calcd for C₃₀H₃₅N₂O₂, 455.27. found 455.2.(6-24)(S)-1-[3-(4-acetylaminophenylsulfanyl)propyl]-3-(carbamoyldiphenylmethyl)-1-methylpyrrolidinium. MS m/z: [M⁺] calcd for C₃₀H₃₆N₃O₂S,502.25. found 502.2.(6-25)(S)-3-(carbamoyldiphenylmethyl)-1-methyl-1-[3-(4-methylsulfanylphenylsulfanyl)propyl]pyrrolidinium. MS m/z: [M⁺] calcd for C₂₉H₃₅N2OS₂,491.22. found 491.2.(6-26)(S)-3-(carbamoyldiphenylmethyl)-1-[3-(4-fluorophenylsulfanyl)propyl]-1-methylpyrrolidinium.MS m/z: [M⁺] calcd for C₂₈H₃₂FN₂OS, 463.22. found 463.6.(6-27)(S)-3-(carbamoyldiphenylmethyl)-1-methyl-1-(3-p-tolylsulfanylpropyl)pyrrolidinium.MS m/z: [M⁺] calcd for C₂₉H₃₅N₂OS, 459.25. found 459.2.(6-28)(S)-3-(carbamoyldiphenylmethyl)-1-methyl-1-[3-(4-methylsulfanylphenoxy)propyl]pyrrolidinium. MS m/z: [M⁺] calcd for C₂₉H₃₅N₂O₂S, 475.24. found475.2.(6-29)(S)-3-(carbamoyldiphenylmethyl)-1-[4-(4-chlorophenyl)-4-oxobutyl]-1-methylpyrrolidinium.MS m/z: [M⁺] calcd for C₂₉H₃₂ClN₂O₂, 475.22. found 475.2.(6-30)(S)-3-(carbamoyldiphenylmethyl)-1-[3-(4-hydroxyphenylsulfanyl)propyl]-1-methylpyrrolidinium.MS m/z: [M⁺] calcd for C₂₈H₃₃N₂O₂S, 461.23. found 461.2.

Example 7

Following the procedures described in the previous examples, andsubstituting the appropriate starting materials and reagents, compounds7-1 to 7-8, having the following formula were also prepared as TFAsalts:

Ex. Q R⁶ 7-1 —(CH₂)₃—O— —CH₃ 7-2 —(CH₂)₃—O— —Br 7-3 —(CH₂)₃—O— —F 7-4—(CH₂)₃—S— —F 7-5 —(CH₂)₃—S— —Cl 7-6 —(CH₂)₃—C(O)— —Cl 7-7 —(CH₂)₃—O——OH 7-8 —(CH₂)₃—S— —OH(7-1)2-[(S)-1-methyl-1-(3-o-tolyloxypropyl)-pyrrolidin-3-yl]-2,2-diphenylacetamide.MS m/z: [M⁺] calcd for C₂₉H₃₅N₂O₂, 443.27. found 443.2.(7-2)2-{(S)-1-[3-(2-bromophenoxy)propyl]-1-methylpyrrolidin-3-yl}-2,2-diphenylacetamide.MS m/z: [M⁺] calcd for C₂₈H₃₂BrN₂O₂, 507.16. found 507.2.(7-3)2-{(S)-1-[3-(2-fluorophenoxy)propyl]-1-methylpyrrolidin-3-yl}-2,2-diphenylacetamide.MS m/z: [M⁺] calcd for C₂₈H₃₂FN₂O₂, 447.24. found 447.2.(7-4)(S)-3-(carbamoyldiphenylmethyl)-1-[3-(2-fluorophenylsulfanyl)propyl]-1-methylpyrrolidinium.MS m/z: [M⁺] calcd for C₂₈H₃₂FN₂OS, 463.22. found 463.2.(7-5)(S)-3-(carbamoyldiphenylmethyl)-1-[3-(2-chlorophenylsulfanyl)propyl]-1-methylpyrrolidinium.MS m/z: [M⁺] calcd for C₂₈H₃₂ClN₂OS, 479.19. found 479.2.(7-6)(S)-3-(carbamoyldiphenylmethyl)-1-[4-(2-chlorophenyl)-4-oxobutyl]-1-methylpyrrolidinium.MS m/z: [M⁺] calcd for C₂₉H₃₂ClN₂O₂, 475.22. found 475.2.(7-7)(S)-3-(carbamoyldiphenylmethyl)-1-[3-(2-hydroxyphenoxy)propyl]-1-methylpyrrolidinium.MS m/z: [M⁺] calcd for C₂₈H₃₃N₂O₃, 445.25. found 445.2.(7-8)(S)-3-(carbamoyldiphenylmethyl)-1-[3-(2-hydroxyphenylsulfanyl)propyl]-1-methylpyrrolidinium.MS m/z: [M⁺] calcd for C₂₈H₃₃N₂O₂S, 461.23. found 461.2.

Example 8

Following the procedures described in the previous examples, andsubstituting the appropriate starting materials and reagents, compounds8-1 to 8-17, having the following formula were also prepared as TFAsalts:

Ex. Q R⁶ 8-1  —(CH₂)₃—O— —CH₃ 8-2  —(CH₂)₃—O— —Cl 8-3  —(CH₂)₃—O— —CN8-4  —(CH₂)₃—S— —Cl 8-5  —(CH₂)₃—S— —F 8-6  —(CH₂)₃—S— —CF₃ 8-7 —(CH₂)₃—S— —OCF₃ 8-8  —CH₂—C(O)NH—CH₂— —F 8-9  —(CH₂)₃—O— —OCH₃ 8-10—(CH₂)₃—O— —F 8-11 —(CH₂)₃—S— —CH₃ 8-12 —(CH₂)₃—C(O)— —OCH₃ 8-13—(CH₂)₃—O— —OH 8-14 —(CH₂)₂—OC(O)— —F 8-15 —(CH₂)₃—S— —OH 8-16—(CH₂)₃—O— —F 8-17 —(CH₂)₂—C≡C— —F(8-1)2-[(S)-1-methyl-1-(3-m-tolyloxypropyl)pyrrolidin-3-yl]-2,2-diphenylacetamide.MS m/z: [M⁺] calcd for C₂₉H₃₅N₂O₂, 443.27. found 443.2.(8-2)2-{(S)-1-[3-(3-chlorophenoxy)propyl]-1-methylpyrrolidin-3-yl}-2,2-diphenylacetamide.MS m/z: [M⁺] calcd for C₂₈H₃₂ClN₂O₂, 463.22. found 463.2.(8-3)2-{(S)-1-[3-(3-cyanophenoxy)propyl]-1-methylpyrrolidin-3-yl}-2,2-diphenylacetamide.MS m/z: [M⁺] calcd for C₂₉H₃₂N₃O₂, 454.25. found 454.2.(8-4)2-{(S)-1-[3-(3-chlorophenylsulfanyl)propyl]-1-methylpyrrolidin-3-yl}-2,2-diphenylacetamide.MS m/z: [M⁺] calcd for C₂₈H₃₂ClN₂OS, 479.19. found 479.2.(8-5)(S)-3-(carbamoyldiphenylmethyl)-1-[3-(3-fluoro-phenylsulfanyl)propyl]-1-methylpyrrolidinium.MS m/z: [M⁺] calcd for C₂₈H₃₂FN₂OS, 463.22. found 463.2. ¹H NMR (CDCl₃,300 MHz) δ (ppm): 1.90 (2H, m), 2.05 (1H, m), 2.14 (2H, br m), 2.62 (3H,s), 2.86 (2H, t), 3.02 (2H, m), 3.22 (1H, s), 3.33 (1H, s), 3.45 (1H,t), 3.59 (1H, d), 3.70 (1H, t), 3.87 (2H, m), 4.18 (1H, m), 5.87 (1H,d), 6.35 (1H, s), 7.0 (4H, m), 7.3 (10H, m).(8-6)(S)-3-(carbamoyldiphenylmethyl)-1-methyl-1-[3-(3-trifluoromethylphenylsulfanyl)propyl]pyrrolidinium. MS m/z: [M⁺] calcd for C₂₉H₃₂F₃N₂OS,513.22. found 513.2.(8-7)(S)-3-(carbamoyldiphenylmethyl)-1-methyl-1-[3-(3-trifluoromethoxyphenylsulfanyl)propyl]pyrrolidinium. MS m/z: [M⁺] calcd for C₂₉H₃₂F₃N₂O₂S,529.21. found 529.2.(8-8)(S)-3-(carbamoyldiphenylmethyl)-1-[(3-fluorobenzylcarbamoyl)methyl]-1-methylpyrrolidinium.MS m/z: [M⁺] calcd for C₂₈H₃₁FN₃O₂, 460.24. found 460.2.(8-9)(S)-3-(carbamoyldiphenylmethyl)-1-[3-(3-methoxyphenoxy)propyl]-1-methylpyrrolidinium.MS m/z: [M⁺] calcd for C₂₉H₃₅N₂O₃, 459.26. found 459.2.(8-10)(S)-3-(carbamoyldiphenylmethyl)-1-[3-(3-fluorophenoxy)propyl]-1-methylpyrrolidinium.MS m/z: [M⁺] calcd for C₂₈H₃₂FN₂O₂, 447.24. found 447.2. ¹H NMR (CDCl₃,300 MHz) δ (ppm): 2.06 (1H, s), 2.18 (2H, s), 2.67 (2H, s), 2.79 (1H,s), 3.26 (3H, s), 3.38 (1H, s), 3.53 (1H, t), 3.68 (1H, s), 3.88 (2H,t), 4.03 (2H, t), 4.16 (1H, m), 5.82 (1H, d), 6.6 (3H, m), 6.51 (1H, s),7.18 (1H, m), 7.3 (10H, m).(8-11)(S)-3-(carbamoyldiphenylmethyl)-1-methyl-1-(3-m-tolylsulfanylpropyl)pyrrolidinium.MS m/z: [M⁺] calcd for C₂₉H₃₅N₂OS, 459.25. found 459.2.(8-12)(S)-3-(carbamoyldiphenylmethyl)-1-[4-(3-methoxyphenyl)-4-oxobutyl]-1-methylpyrrolidinium.MS m/z: [M⁺] calcd for C₃₀H₃₅N₂O₃, 471.26. found 471.2.(8-13)(S)-3-(carbamoyldiphenylmethyl)-1-[3-(3-hydroxyphenoxy)propyl]-1-methylpyrrolidinium.MS m/z: [M⁺] calcd for C₂₈H₃₃N₂O₃, 445.25. found 445.2.(8-14)(S)-3-(carbamoyldiphenylmethyl)-1-[2-(3-fluorobenzoyloxy)ethyl]-1-methylpyrrolidinium.MS m/z: [M⁺] calcd for C₂₈H₃₀FN₂O₃, 461.22. found 461.2.(8-15)(S)-3-(carbamoyldiphenylmethyl)-1-[3-(3-hydroxyphenylsulfanyl)propyl]-1-methylpyrrolidinium.MS m/z: [M⁺] calcd for C₂₈H₃₃N₂O₂S, 461.23. found 461.2.(8-16)(R)-3-(carbamoyldiphenylmethyl)-1-[3-(3-fluorophenoxy)propyl]-1-methylpyrrolidinium.MS m/z: [M⁺] calcd for C₂₈H₃₂FN₂O₂, 448.24. found 447.2.(8-17)(S)-3-(carbamoyldiphenylmethyl)-1-[4-(3-fluorophenyl)but-3-ynyl]-1-methylpyrrolidinium.MS m/z: [M⁺] calcd for C₂₉H₃₀FN₂O, 442.23. found 441.2.

Example 9

Following the procedures described in the previous examples, andsubstituting the appropriate starting materials and reagents, compounds9-1 to 9-10, having the following formula were also prepared as TFAsalts:

Ex. Q 9-1  —(CH₂)₃—S— 9-2  —(CH₂)₂—NHC(O)NH— 9-3  —CH₂—C(O)O—CH₂— 9-4 —(CH₂)₃—O—CH₂— 9-5  —(CH₂)₃—S(O)— 9-6  —(CH₂)₃—SO₂— 9-7  —(CH₂)₃—S— 9-8 —(CH₂)₃—O— 9-9  —(CH₂)₂—C≡C— 9-10 —(CH₂)₃—O—(9-1)(S)-3-(carbamoyldiphenylmethyl)-1-[3-(3,5-difluorophenylsulfanyl)propyl]-1-methylpyrrolidinium.MS m/z: [M⁺] calcd for C₂₈H₃₁F₂N₂OS, 481.21. found 481.2. ¹H NMR (CDCl₃,300 MHz) δ (ppm): 2.02 (3H, m), 2.63 (s, 3H), 2.78 (1H, s), 2.88 (1H,s), 3.05 (1H, m), 3.24 (1H, s), 3.34 (1H, s), 3.48 (1H, s), 3.61 (1H,s), 3.72 (1H, s), 3.86 (2H, m), 4.14 (1H, m), 5.80 (2H, d), 6.22 (1H,s), 6.62 (1H, q), 6.79 (2H, dd), 7.35 (10H, m).(9-2)(S)-3-(carbamoyldiphenylmethyl)-1-{2-[3-(3,5-difluorophenyl)ureido]ethyl}-1-methylpyrrolidinium.MS m/z: [M⁺] calcd for C₂₈H₃₁F₂N₄O₂, 493.24. found 493.2.(9-3)(S)-3-(carbamoyldiphenylmethyl)-1-(3,5-difluorobenzyloxycarbonylmethyl)-1-methylpyrrolidinium.MS m/z: [M⁺] calcd for C₂₈H₂₉F₂N₂O₃, 479.21. found 479.2.(9-4)(S)-3-(carbamoyldiphenylmethyl)-1-[3-(3,5-difluorobenzyloxy)propyl]-1-methylpyrrolidinium.MS m/z: [M⁺] calcd for C₂₉H₃₃F₂N₂O₂, 479.25. found 479.2.(9-5)(S)-3-(Carbamoyldiphenylmethyl)-1-[3-(3,5-difluorobenzenesulfinyl)propyl]-1-methylpyrrolidinium.MS m/z: [M⁺] calcd for C₂₈H₃₁F₂N₂O₂S, 497.21. found 497.2.(9-6)(S)-3-(Carbamoyldiphenylmethyl)-1-[3-(3,5-difluorobenzenesulfonyl)propyl]-1-methyl-pyrrolidinium.MS m/z: [M⁺] calcd for C₂₈H₃₁F₂N₂O₃S, 513.20. found 513.2.(9-7)(1S,3S)-3-(carbamoyldiphenylmethyl)-1-[3-(3,5-difluorophenylsulfanyl)propyl]-1-methylpyrrolidinium. MS m/z: [M⁺] calcd for C₂₈H₃₁F₂N₂OS,482.21. found 482.2.(9-8)(1S,3S)-3-(carbamoyldiphenylmethyl)-1-[3-(3,5-difluorophenoxy)propyl]-1-methylpyrrolidinium.MS m/z: [M⁺] calcd for C₂₈H₃₁F₂N₂O₂, 466.24. found 465.2.(9-9)(S)-3-(carbamoyldiphenylmethyl)-1-[4-(3,5-difluorophenyl)but-3-ynyl]-1-methylpyrrolidinium.MS m/z: [M⁺] calcd for C₂₉H₂₉F₂N₂O, 460.22. found 459.2.(9-10)(R)-3-(carbamoyldiphenylmethyl)-1-[3-(3,5-difluorophenoxy)propyl]-1-methylpyrrolidinium.MS m/z: [M⁺] calcd for C₂₈H₃₁F₂N₂O₂, 466.24. found 465.2.

Preparation 6 (3-Methoxyphenyl)phenylacetonitrile

A solution of 1-(chlorophenylmethyl)-3-methoxybenzene (5.5 g, 24 mmol)in DCM (100 mL) was cooled to 0° C. Trimethylsilyl cyanide (3.5 mL, 26mmol) was added to the stirred solution. 1.0 M of tin tetrachloride inheptane (0.75 mL, 0.75 mmol) was then added. The solution was stirredfor 2 hours, then quenched with MeOH (50 mL). The solvent was removedunder reduced pressure. The crude material was then purified via silicagel chromatography to obtain the title compound (2.2 g, 10 mmol).

Preparation 7((S)-1-Benzlpyrrolidin-3-yl)(3-methoxyphenyl)phenylacetonitrile

To a stirred solution of (3-methoxyphenyl)phenylacetonitrile (5.2 g, 23mmol; prepared as described in Preparation 6) in THF (100 mL) was addedtoluene-4-sulfonic acid (S)-1-benzylpyrrolidin-3-yl ester (7.2 g, 22mmol), followed by the addition of potassium t-butoxide (4.0 g, 36mmol). The mixture was heated to 80° C. for 1 hour, then allowed to cooland H₂O (10 mL) was added. The mixture was filtered through a pad ofCelite®. The mixture was washed with a saturated aqueous NaCl solution(100 mL) and the organic phase was separated, dried over MgSO₄, andfiltered. The solvent was removed under reduced pressure. The crudematerial was purified via silica gel chromatography to afford the titlecompound (6.2 g, 17 mmol). MS m/z: {M+H+] calcd for C₂₆H₂₆N₂O, 382.5.found 383.4.

Preparation 82-(3-Hydroxyphenyl)-2-phenyl-2-(S)-pyrrolidin-3-yl-acetamide

To a stirred solution of((S)-1-benzylpyrrolidin-3-yl)-(3-methoxyphenyl)phenylacetonitrile (6.2g, 16 mmol; prepared as described in Preparation 7) in t-butyl alcohol(150 mL) was added KOH (35 g, 620 mmol). The reaction flask was equippedwith a reflux condenser and nitrogen inlet and stirred at 110° C. for 14days. The mixture was allowed to cool to room temperature. Water (120mL) and ether (100 mL) were added to the mixture. The organic phase wasthen separated, and the aqueous phase was extracted 2 times with ether(100 mL each). The organic layers were combined, dried over MgSO₄, thenfiltered. The solvent was removed under reduced pressure and the crudematerial was then taken in DCM (250 mL) and cooled to 0° C. To thisstirred solution was added 1.0 M of boron tribromide in DCM (30 mL, 30mmol) and the mixture was stirred at 0° C. for 90 minutes. The mixturewas then added to a stirred solution of 30% ammonia & ice. The mixturewas then filtered through a pad of Celite®, then washed with a saturatedaqueous NaCl solution (200 mL). The organic phase was dried over MgSO₄,then filtered. The solvent was removed under reduced pressure. The crudematerial was then subjected to normal hydrogenation conditions to obtainthe title compound. MS m/z: {M+H+] calcd for C₁₈H₂₀N₂O₂, 296.36. found297.6.

This material was used to prepare the compound of Example 10-3.

Example 10

Following the procedures described in the previous examples, andsubstituting the appropriate starting materials and reagents, compounds10-1 to 10-3, having the following formula were also prepared as TFAsalts:

Ex. a R¹ b R² 10-1 1 4-hydroxy 1 4-hydroxy 10-2 1 4-hydroxy 0 — 10-3 13-hydroxy 0 —(10-1)(S)-3-[carbamoyl-bis-(4-hydroxyphenyl)methyl]-1-[3-(3,5-difluorophenylsulfanyl)propyl]-1-methylpyrrolidinium.MS m/z: [M⁺] calcd for C₂₈H₃₁F₂N₂O₃S, 514.20. found 513.2.(10-2)(S)-3-[(S)-carbamoyl-(4-hydroxyphenyl)phenylmethyl]-1-[3-(3,5-difluorophenylsulfanyl)propyl]-1-methylpyrrolidinium.MS m/z: [M⁺] calcd for C₂₈H₃₁F₂N₂O₂S, 498.21. found 497.2.(10-3)(S)-3-[carbamoyl-(3-hydroxyphenyl)phenylmethyl]-1-[3-(3,5-difluorophenylsulfanyl)propyl]-1-methylpyrrolidinium.MS m/z: [M⁺] calcd for C₂₈H₃₁F₂N₂O₂S, 498.21. found 497.2.

Example 11

Following the procedures described in the previous examples, andsubstituting the appropriate starting materials and reagents, compoundsII-1 to 11-3, having the following formula were also prepared as TFAsalts:

Ex. Q R^(6a) R^(6b) 11-1 —(CH₂)₃—S— —F —OCH₃ 11-2 —(CH₂)₃—O— —F —F 11-3—(CH₂)₃—S— —F —F(11-1)(S)-3-(carbamoyldiphenylmethyl)-1-[3-(3-fluoro-4-methoxyphenylsulfanyl)propyl]-1-methylpyrrolidinium. MS m/z: [M⁺] calcd for C₂₉H₃₄FN₂O₂S,493.23. found 493.2.(11-2)(S)-3-(carbamoyldiphenylmethyl)-1-[3-(3,4-difluorophenoxy)propyl]-1-methylpyrrolidinium.MS m/z: [M⁺] calcd for C₂₈H₃₁F₂N₂O₂, 465.24. found 465.2.(11-3)(S)-3-(carbamoyldiphenylmethyl)-1-[3-(3,4-difluorophenylsulfanyl)propyl]-1-methylpyrrolidinium.MS m/z: [M⁺] calcd for C₂₈H₃₁F₂N₂OS, 481.21. found 481.2.

Example 12

Following the procedures described in the previous examples, andsubstituting the appropriate starting materials and reagents, compounds12-1 to 12-2, having the following formula were also prepared as TFAsalts:

Ex. Q 12-1 —(CH₂)₃—O— 12-2 —(CH₂)₂—OC(O)—(12-1)(S)-3-(carbamoyldiphenylmethyl)-1-[3-(2,3-difluorophenoxy)propyl]-1-methylpyrrolidinium.MS m/z: [M⁺] calcd for C₂₈H₃₁F₂N₂O₂, 465.24. found 465.2.(12-2)(S)-3-(carbamoyldiphenylmethyl)-1-[2-(2,3-difluorobenzoyloxy)ethyl]-1-methylpyrrolidinium.MS m/z: [M⁺] calcd for C₂₈H₂₉F₂N₂O₃, 479.21. found 479.2.

Example 13

Following the procedures described in the previous examples, andsubstituting the appropriate starting materials and reagents, compounds13-1 to 13-2, having the following formula were also prepared as TFAsalts:

Ex. Q 13-1 —(CH₂)₃—O— 13-2 —(CH₂)₃—S—(13-1)(S)-3-(carbamoyldiphenylmethyl)-1-[3-(2,4-difluorophenoxy)propyl]-1-methylpyrrolidinium.MS m/z: [M⁺] calcd for C₂₈H₃₁F₂N₂O₂, 465.24. found 465.2.(13-2)(S)-3-(carbamoyldiphenylmethyl)-1-[3-(2,4-difluorophenylsulfanyl)propyl]-1-methylpyrrolidinium.MS m/z: [M⁺] calcd for C₂₈H₃₁F₂N₂OS, 481.21. found 481.2.

Example 14

Following the procedures described in the previous examples, andsubstituting the appropriate starting materials and reagents, compound14, having the following formula was also prepared as a TFA salt:

(S)-3-(carbamoyldiphenylmethyl)-1-[3-(2,6-difluorophenoxy)propyl]-1-methylpyrrolidinium.MS m/z: [M⁺] calcd for C₂₈H₃₁F₂N₂O₂, 465.24. found 465.2.

Example 15

Following the procedures described in the previous examples, andsubstituting the appropriate starting materials and reagents, compound15, having the following formula was also prepared as a TFA salt:

(S)-3-(carbamoyldiphenylmethyl)-1-[2-(2,5-difluorobenzoyloxy)ethyl]-1-methylpyrrolidinium.MS m/z: [M⁺] calcd for C₂₈H₂₉F₂N₂O₃, 479.21. found 479.2.

Example 16

Following the procedures described in the previous examples, andsubstituting the appropriate starting materials and reagents, compounds16-1 to 16-3, having the following formula were also prepared as TFAsalts:

(16-1)(S)-3-(carbamoylphenyl-m-tolylmethyl)-1-methyl-1-(4-phenylbutyl)pyrrolidinium(R¹=3-methyl). MS m/z: [M⁺] calcd for C₃₀H₃₇N₂O, 441.29. found 441.2.(16-2)(S)-3-(carbamoylphenyl-p-tolylmethyl)-1-methyl-1-(4-phenylbutyl)pyrrolidinium(R¹=4-methyl). MS m/z: [M⁺] calcd for C₃₀H₃₇N₂O, 441.29. found 441.2.(16-3)(S)-3-[carbamoyl(4-hydroxyphenyl)phenylmethyl]-1-methyl-1-(4-phenylbutyl)pyrrolidinium(R¹=4-hydroxy). MS m/z: [M⁺] calcd for C₂₉H₃₅N₂O₂, 443.27. found 443.2.

Assay 1 Radioligand Binding Assay Membrane Preparation from CellsExpressing hM₁, hM₂, hM₃ and hM₄ Muscarinic Receptor Subtypes

CHO cell lines stably expressing cloned human hM₁, hM₂, hM₃ and hM₄muscarinic receptor subtypes, respectively, were grown to nearconfluency in medium consisting of HAM's F-12 supplemented with 10% FBSand 250 μg/mL Geneticin. The cells were grown in a 5% CO₂, 37° C.incubator and lifted with 2 mM EDTA in dPBS. Cells were collected by 5minute centrifugation at 650×g, and cell pellets were either storedfrozen at −80° C. or membranes were prepared immediately. For membranepreparation, cell pellets were resuspended in lysis buffer andhomogenized with a Polytron PT-2100 tissue disrupter (Kinematica AG; 20seconds×2 bursts). Crude membranes were centrifuged at 40,000×g for 15minutes at 4° C. The membrane pellet was then resuspended withresuspension buffer and homogenized again with the Polytron tissuedisrupter. The protein concentration of the membrane suspension wasdetermined by the method described in Lowry, O. et al., Journal ofBiochemistry 193:265 (1951). All membranes were stored frozen inaliquots at −80° C. or used immediately. Aliquots of prepared hM₅receptor membranes were purchased directly from Perkin Elmer and storedat −80° C. until use.

Radioligand Binding Assay on Muscarinic Receptor Subtypes hM₁, hM₂, hM₃,hM₄ and hM₅

Radioligand binding assays were performed in 96-well microtiter platesin a total assay volume of 1000 μL. CHO cell membranes stably expressingeither the hM₁, hM₂, hM₃, hM₄ or hM₅ muscarinic subtype were diluted inassay buffer to the following specific target protein concentrations(g/well): 10 μg for hM₁, 10-15 μg for hM₂, 10-20 μg for hM₃, 10-20 μgfor hM₄, and 10-12 μg for hM₅. The membranes were briefly homogenizedusing a Polytron tissue disruptor (10 seconds) prior to assay plateaddition. Saturation binding studies for determining K_(D) values of theradioligand were performed using L-[N-methyl-³H]scopolamine methylchloride ([³H]-NMS) (TRK666, 84.0 Ci/mmol, Amersham Pharmacia Biotech,Buckinghamshire, England) at concentrations ranging from 0.001 nM to 20nM. Displacement assays for determination of K_(i) values of testcompounds were performed with [³H]-NMS at 1 nM and eleven different testcompound concentrations. The test compounds were initially dissolved toa concentration of 40 μM in dilution buffer and then serially diluted 5×with dilution buffer to final concentrations ranging from 400 fM to 4μM. The addition order and volumes to the assay plates were as follows:825 μL assay buffer with 0.1% BSA, 25 μL radioligand, 100 μL dilutedtest compound, and 50 μL membranes. Assay plates were incubated for 6hours at 37° C. Binding reactions were terminated by rapid filtrationover GF/B glass fiber filter plates (Perkin Elmer Inc., Wellesley,Mass.) pre-treated in 0.3% polyethyleneimine (PEI). Filter plates wererinsed three times with wash buffer (10 mM HEPES) to remove unboundradioactivity. Plates were then air dried, and 50 μL Microscint-20liquid scintillation fluid (PerkinElmer Inc., Wellesley, Mass.) wasadded to each well. The plates were then counted in a PerkinElmerTopcount liquid scintillation counter (PerkinElmer Inc., Wellesley,Mass.). Binding data were analyzed by nonlinear regression analysis withthe GraphPad Prism Software package (GraphPad Software, Inc., San Diego,Calif.) using the one-site competition model. K_(i) values for testcompounds were calculated from observed IC₅₀ values and the K_(D) valueof the radioligand using the Cheng-Prusoff equation (Cheng Y; Prusoff W.H. Biochemical Pharmacology 22(23):3099-108 (1973)). K_(i) values wereconverted to pK_(i) values to determine the geometric mean and 95%confidence intervals. These summary statistics were then converted backto K_(i) values for data reporting.

In this assay, a lower K_(i) value indicates that the test compound hasa higher binding affinity for the receptor tested. Exemplary compoundsof the invention that were tested in this assay, were found to have aK_(i) value of less than about 120 nM for the M₃ muscarinic receptorsubtype in this assay. More typically, these compounds were found tohave K_(i) values of less than about 50 nM, with some compounds havingK_(i) values of less than about 10 nM or less than about 1.0 nM.

Assay 2 Muscarinic Receptor Functional Potency Assays Blockade ofAgonist-Mediated Inhibition of cAMP Accumulation

In this assay, the functional potency of a test compound is determinedby measuring the ability of the test compound to blockoxotremorine-inhibition of forskolin-mediated cAMP accumulation inCHO-K1 cells expressing the hM₂ receptor.

cAMP assays are performed in a radioimmunoassay format using theFlashplate Adenylyl Cyclase Activation Assay System with ¹²⁵I-cAMP (NENSMP004B, PerkinElmer Life Sciences Inc., Boston, Mass.), according tothe manufacturer's instructions.

Cells are rinsed once with dPBS and lifted with Trypsin-EDTA solution(0.05% trypsin/0.53 mM EDTA) as described in Assay 1. The detached cellsare washed twice by centrifugation at 650×g for five minutes in 50 mLsdPBS. The cell pellet is then re-suspended in 10 mL dPBS, and the cellsare counted with a Coulter Z1 Dual Particle Counter (Beckman Coulter,Fullerton, Calif.). The cells are centrifuged again at 650×g for fiveminutes and re-suspended in stimulation buffer to an assay concentrationof 1.6×10⁶-2.8×10⁶ cells/mL.

The test compound is initially dissolved to a concentration of 400 M indilution buffer (dPBS supplemented with 1 mg/mL BSA (0.1%)), and thenserially diluted with dilution buffer to final molar concentrationsranging from 100 μM to 0.1 nM. Oxotremorine is diluted in a similarmanner.

To measure oxotremorine inhibition of AC activity, 25 μL forskolin (25 Mfinal concentration diluted in dPBS), 25 μL diluted oxotremorine, and 50μL cells are added to agonist assay wells. To measure the ability of atest compound to block oxotremorine-inhibited AC activity, 25 μLforskolin and oxotremorine (25 μM and 5 μM final concentrations,respectively, diluted in dPBS) 25 μL diluted test compound, and 50 μLcells are added to remaining assay wells.

Reactions are incubated for 10 minutes at 37° C. and stopped by additionof 100 μL ice-cold detection buffer. Plates are sealed, incubatedovernight at room temperature and counted the next morning on aPerkinElmer TopCount liquid scintillation counter (PerkinElmer Inc.,Wellesley, Mass.). The amount of cAMP produced (pmol/well) is calculatedbased on the counts observed for the samples and cAMP standards, asdescribed in the manufacturer's user manual. Data are analyzed bynonlinear regression analysis with the GraphPad Prism Software package(GraphPad Software, Inc., San Diego, Calif.) using the non-linearregression, one-site competition equation. The Cheng-Prusoff equation isused to calculate the K_(i), using the EC₅₀ of the oxotremorineconcentration-response curve and the oxotremorine assay concentration asthe K_(D) and [L], respectively. The K_(i) values are converted topK_(i) values to determine the geometric mean and 95% confidenceintervals. These summary statistics are then converted back to K_(i)values for data reporting.

In this assay, a lower K_(i) value indicates that the test compound hasa higher functional activity at the receptor tested. The exemplifiedcompounds of the invention are expected to have a K_(i) value of lessthan about 100 nM for blockade of oxotremorine-inhibition offorskolin-mediated cAMP accumulation in CHO-K1 cells expressing the hM₂receptor.

Blockade of Agonist-Mediated [³⁵S]GTPγS Binding

In a second functional assay, the functional potency of test compoundscan be determined by measuring the ability of the compounds to blockoxotremorine-stimulated [³⁵S]GTPγS binding in CHO-K1 cells expressingthe hM₂ receptor.

At the time of use, frozen membranes are thawed and then diluted inassay buffer with a final target tissue concentration of 5-10 μg proteinper well. The membranes are briefly homogenized using a Polytron PT-2100tissue disrupter and then added to the assay plates.

The EC₉₀ value (effective concentration for 90% maximal response) forstimulation of [³⁵S]GTPγS binding by the agonist oxotremorine isdetermined in each experiment.

To determine the ability of a test compound to inhibitoxotremorine-stimulated [³⁵S]GTPγS binding, the following is added toeach well of 96 well plates: 25 μL of assay buffer with [³⁵S]GTPγS (0.4nM), 25 μL of oxotremorine (EC₉₀) and GDP (3 μM), 25 μL of diluted testcompound and 25 μL CHO cell membranes expressing the hM₂ receptor. Theassay plates are then incubated at 37° C. for 60 minutes. The assayplates are filtered over 1% BSA-pretreated GF/B filters using aPerkinElmer 96-well harvester. The plates are rinsed with ice-cold washbuffer for 3×3 seconds and then air or vacuum dried. Microscint-20scintillation liquid (50 μL) is added to each well, and each plate issealed and radioactivity counted on a topcounter (PerkinElmer). Data areanalyzed by nonlinear regression analysis with the GraphPad PrismSoftware package (GraphPad Software, Inc., San Diego, Calif.) using thenon-linear regression, one-site competition equation. The Cheng-Prusoffequation is used to calculate the K_(i), using the IC₅₀ values of theconcentration-response curve for the test compound and the oxotremorineconcentration in the assay as the K_(D) and [L], ligand concentration,respectively.

In this assay, a lower K_(i) value indicates that the test compound hasa higher functional activity at the receptor tested. The exemplifiedcompounds of the invention are expected to have a K_(i) value of lessthan about 100 nM for blockade of oxotremorine-stimulated [³⁵S]GTPγSbinding in CHO-K1 cells expressing the hM₂ receptor.

Blockade of Agonist-Mediated Calcium Release Via FLIPR Assays

Muscarinic receptor subtypes (M₁, M₃ and M₅ receptors), which couple toG_(q) proteins, activate the phospholipase C (PLC) pathway upon agonistbinding to the receptor. As a result, activated PLC hydrolyzesphosphatyl inositol diphosphate (PIP₂) to diacylglycerol (DAG) andphosphatidyl-1,4,5-triphosphate (IP₃), which in turn generates calciumrelease from intracellular stores, i.e., endoplasmic and sarcoplasmicreticulum. The FLIPR (Molecular Devices, Sunnyvale, Calif.) assaycapitalizes on this increase in intracellular calcium by using a calciumsensitive dye (Fluo-4AM, Molecular Probes, Eugene, Oreg.) thatfluoresces when free calcium binds. This fluorescence event is measuredin real time by the FLIPR, which detects the change in fluorescence froma monolayer of cells cloned with human M₁ and M₃, and chimpanzee M₅receptors. Antagonist potency can be determined by the ability ofantagonists to inhibit agonist-mediated increases in intracellularcalcium.

For FLIPR calcium stimulation assays, CHO cells stably expressing thehM₁, hM₃ and cM₅ receptors are seeded into 96-well FLIPR plates thenight before the assay is done. Seeded cells are washed twice byCellwash (MTX Labsystems, Inc.) with FLIPR buffer (10 mM HEPES, pH 7.4,2 mM calcium chloride, 2.5 mM probenecid in HBSS without calcium andmagnesium) to remove growth media and leaving 50 μL/well of FLIPRbuffer. The cells are then incubated with 50 μL/well of 4 μM FLUO-4AM (a2× solution was made) for 40 minutes at 37° C., 5% carbon dioxide.Following the dye incubation period, cells are washed two times withFLIPR buffer, leaving a final volume of 50 μL/well.

To determine antagonist potency, the dose-dependent stimulation ofintracellular Ca²⁺ release for oxotremorine is first determined so thatantagonist potency can later be measured against oxotremorinestimulation at an EC₉₀ concentration. Cells are first incubated withcompound dilution buffer for 20 minutes, followed by agonist addition,which is performed by the FLIPR. An EC₉₀ value for oxotremorine isgenerated according to the method detailed in the FLIPR measurement anddata reduction section below, in conjunction with the formulaEC_(F)=((F/100−F)^1/H)*EC₅₀. An oxotremorine concentration of 3×EC_(F)is prepared in stimulation plates such that an EC₉₀ concentration ofoxotremorine is added to each well in the antagonist inhibition assayplates.

The parameters used for the FLIPR are: exposure length of 0.4 seconds,laser strength of 0.5 watts, excitation wavelength of 488 nm, andemission wavelength of 550 nm. Baseline is determined by measuring thechange in fluorescence for 10 seconds prior to addition of agonist.Following agonist stimulation, the FLIPR continuously measures thechange of fluorescence every 0.5 to 1 second for 1.5 minutes to capturethe maximum fluorescence change.

The change of fluorescence is expressed as maximum fluorescence minusbaseline fluorescence for each well. The raw data is analyzed againstthe logarithm of drug concentration by nonlinear regression withGraphPad Prism (GraphPad Software, Inc., San Diego, Calif.) using thebuilt-in model for sigmoidal dose-response. Antagonist K_(i) values aredetermined by Prism using the oxotremorine EC₅₀ value as the K_(D) andthe oxotremorine EC₉₀ for the ligand concentration according to theCheng-Prusoff equation (Cheng & Prusoff, 1973).

In this assay, a lower K_(i) value indicates that the test compound hasa higher functional activity at the receptor tested. The exemplifiedcompounds of the invention are expected to have a K_(i) value of lessthan about 100 nM for blockade of agonist-mediated calcium release inCHO cells stably expressing the hM₃ receptor.

Assay 3 Rat Einthoven Assay

This in vivo assay is used to assess the bronchoprotective effects oftest compounds exhibiting muscarinic receptor antagonist activity.

All test compounds are diluted in sterile water and are dosed via theinhalation route (IH). The rats (Sprague-Dawley, male, 250-350 g) areexposed to the aerosol generated from an LC Star Nebulizer Set anddriven by a mixture of gases (5% CO₂/95% atmospheric air). Each testcompound solution is nebulized over a 10 minute time period in a pieshaped dosing chamber capable of holding six rats. At predetermined timepoints after inhalation of compound, the Einthoven assay is performed.

Thirty minutes prior to the start of pulmonary evaluation, the animalsare anesthetized with inactin (thiobutabarbital, 120 mg/kg IP). Thejugular vein is catheterized with saline filled polyethylene catheters(PE-50) and used to infuse MCh. The trachea is then dissected andcannulated with a 14G needle and used for rat ventilation duringpulmonary evaluation. Once surgery is complete, rats are ventilatedusing a piston respirator set at a stroke volume of 1 ml/100 g bodyweight but not exceeding 2.5 ml volume, and at a rate of 90 strokes perminute.

The changes in pressure that occur with each breath are measured.Baseline values are collected for at least 2.5 minutes then rats arechallenged non-cumulatively with 2-fold incremental increases of thebronchoconstrictor MCh (5, 10, 20, 40 and 80 μg/ml). The MCh is infusedfor 2.5 minutes from a syringe pump at a rate of 2 mL/kg/min. Theanimals are euthanized upon completion of the studies.

Changes in ventilation pressure (cm H₂O) in treated animals areexpressed as % inhibition of MCh response relative to control animals.In this assay, a higher % inhibition value indicates that the testcompound has a bronchoprotective effect. Exemplary compounds of theinvention that are tested in this assay at a dose of 100 g/ml areexpected to exhibit greater than 35% inhibition, some are expected toexhibit greater than 70% inhibition, and some are expected to exhibitgreater than 90% inhibition.

1.5 hr ID₅₀ Determination

Standard muscarinic antagonists were evaluated in the rat Einthovenassay 1.5 hrs post-dose. The order of potency (ID₅₀s) for the fivestandards tested was determined to be: ipratropium (4.4μg/ml)>tiotropium (6 μg/ml)>des-methyl-tiotropium (12μg/ml)>glycopyrrolate (15 μg/ml)>LAS-34237 (24 μg/ml). The potency ofthe test compound is similarly determined at 1.5 hrs post-dose.

6 and 24 hr ID₅₀ Determination

Standards tiotropium and ipratropium were also evaluated 24 hr and/or 6hr post-dose in the rat Einthoven assay. Ipratropium (10 and 30 μg/ml)was about 3-fold less potent 6-hr post-dose compared to its 1.5 hrpotency. The observed loss of activity at this time point (6 hr) isconsistent with its relatively short duration of action in the clinic.Tiotropium showed a slow onset of effect with peak bronchoprotectionbeing achieved 6-hr post-dose. Its 6 hr and 24 hr potency values werenot significantly different from each other and were about 2-fold morepotent compared to its 1.5 hr potency. The onset of action of the testcompound, as well as the 6 and 24 hr potency values, is similarlydetermined.

Assay 4 Rat Antisialagogue Assay

Rats (Sprague-Dawley, male, 250-350 g) are dosed, anesthetized andcannulated as described for Assay 3. At predetermined time points andafter surgery, animals are placed on their dorsal side at a 200 inclinewith their head in a downward slope. A pre-weighed gauze pad is insertedin the animal's mouth and the muscarinic agonist pilocarpine (PILO) (3mg/kg, iv.) is administered. Saliva produced during 10 minutes post-PILOis measured gravimetrically by determining the weight of the gauze padbefore and after PILO. Antisialagogue effects are expressed as %inhibition of salivation relative to control animals.

1, 6 and 24 hr ID₅₀ Determination

The rat antisialagogue assay was developed to assess systemic exposureand calculate the lung selectivity index (LSI) of test compounds. Thestandard, tiotropium, was evaluated in this model at 1, 6, and 24 hrpost-dose. Tiotropium was found to be most potent at inhibitingpilocarpine-induced salivation 6 hrs post dose. This finding isconsistent with the peak effects observed in the Einthoven assay.

This model is a modified version of the procedure described in Rechter,“Estimation of anticholinergic drug effects in mice by antagonismagainst pilocarpine-induced salivation” Ata Pharmacol Toxicol 24:243-254(1996). The mean weight of saliva in vehicle-treated animals, at eachpre-treatment time, is calculated and used to compute % inhibition ofsalivation, at the corresponding pre-treatment time, at each dose.

Exemplary compounds of the invention that are tested in this assay areexpected to exhibit ID₅₀ values less than 100 μg/ml (measured at 24hours), with some compounds expected to exhibit an ID₅₀ value less than30 μg/ml, some less than 20 μg/ml, and some less than 15 μg/ml.

The ratio of the anti-sialagogue ID₅₀ to bronchoprotective ID₅₀ is usedto compute the apparent lung selectivity index of the test compound.Generally, compounds having an apparent lung selectivity index greaterthan about 5 are preferred.

While the present invention has been described with reference tospecific aspects or embodiments thereof, it will be understood by thoseof ordinary skilled in the art that various changes can be made orequivalents can be substituted without departing from the true spiritand scope of the invention. Additionally, to the extent permitted byapplicable patent statues and regulations, all publications, patents andpatent applications cited herein are hereby incorporated by reference intheir entirety to the same extent as if each document had beenindividually incorporated by reference herein.

What is claimed is:
 1. A process for preparing a compound of formula I:

in salt or zwitterionic form, wherein: a and b are independently 0 or aninteger of from 1 to 5; each R^(l) and R² is independently selected from—C₁₋₄alkyl, —C₂₋₄alkenyl, —C₂₋₄alkynyl, —C₃₋₆cycloalkyl, cyano, halo,—OR^(a), —CH₂OH, —COOH, —C(O)—O—C₁₋₄alkyl, —C(O)NR^(b)R^(c), —SR^(a),—S(O)R^(a), —S(O)₂R^(a), and —NR^(b)R^(c); where each R^(a) isindependently selected from hydrogen, —C₁₋₄alkyl, —C₂₋₄alkenyl,—C₂₋₄alkynyl and —C₃₋₆cycloalkyl; each R^(b) and R^(c) is independentlyselected from hydrogen, —C₁₋₄alkyl, —C₂₋₄alkenyl, —C₂₋₄alkynyl, and—C₃₋₆cycloalkyl; or R^(b) and R^(c) together with the nitrogen atom towhich they are attached form a C₃₋₆heterocycle; or two adjacent R^(l)groups or two adjacent R² groups are joined together to form—C₃₋₆alkylene, —C₂₋₄alkylene-O— or —O—C₁₋₄alkylene-O—; R³ is selectedfrom —C(O)NR^(3a)R^(3b), —C(O)O—C₁₋₄alkyl, —CN, —CH₂OH, and —CH₂NH₂;where R^(3a) and R^(3b) are independently selected from hydrogen,—C₁₋₆alkyl, —C₂₋₆alkenyl, —C₂₋₆alkynyl, —C₃₋₆cycloalkyl, —C₆₋₁₀aryl,—C₂₋₉heteroaryl, —C₃₋₆heterocycle, and —(CH₂)₁₋₂—R^(3c), where R^(3c) isselected from —OH, —O—C₁₋₆alkyl, —C₃₋₆cycloalkyl, —C₆₋₁₀aryl,—C₂₋₉heteroaryl, and —C₃₋₆heterocycle; or R^(3a) and R^(3b) togetherwith the nitrogen atom to which they are attached form a C₃₋₆heterocycleoptionally containing one additional heteroatom selected from nitrogen,oxygen, and sulfur; c is 0 or an integer of from 1 to 3; each R⁴ isindependently fluoro or —C₁₋₄alkyl; d is 1 or 2, and depicts an optionaldouble bond; R⁵ is selected from —C₁₋₆alkyl, —CH₂—C₂₋₆alkenyl,—CH₂—C₂₋₆alkynyl, and —CH₂COR^(5a); where R^(5a) is selected from —OH,—O—C₁₋₆alkyl, and —NR^(5b)R^(5c); and R^(5b) and R^(5a) areindependently selected from H and —C₁₋₆alkyl; Q is selected from—C₀₋₅alkylene-Q′-C₀₋₁alkylene-, wherein Q′ is selected from —CH₂—,—CH═CH—, —C≡C—, —O—, —S—, —S(O)—, —SO₂—, —SO₂—NR^(Q1)-, —NR^(Q1)—SO₂—,—C(O)—, —OC(O)—, —C(O)O—, —NR^(Q1)C(O)—, —C(O)NR^(Q1)-,NR^(Q2)—C(O)—NR^(Q3)-, —NR^(Q2)—C(S)—NR^(Q3)-, —C═N—O—, —S—S—, and—C(═N—O—R^(Q4))—, where R^(Q1) is hydrogen or —C₁₋₄alkyl, R^(Q2) andR^(Q3) are independently selected from hydrogen, —C₁₋₄alkyl, and—C₃₋₆cycloalkyl, or R^(Q2) and R^(Q3) are taken together to form—C₂₋₄alkylene or —C₂₋₃alkenylene, and R^(Q4) is —C₁₋₄alkyl or benzyl; eis 0 or an integer of from 1 to 5; each R⁶ is independently selectedfrom halo, —C₁₋₄alkyl, —C₀₋₄alkylene-OH, cyano, —C₀₋₂alkylene-COOH,—C(O)O—C₁₋₄alkyl, —O—C₁₋₄alkyl, —S—C₁₋₄alkyl, —NH—C(O)—C₁₋₄alkyl,—N(C₁₋₄alkyl)₂, and —N⁺(O)O; wherein each alkyl, alkenyl, alkylene,alkynyl and cycloalkyl group in R¹⁻³, R^(3a-3c), R⁴⁻⁶, and R^(a-c) isoptionally substituted with 1 to 5 fluoro atoms; wherein each alkyl,alkenyl, and alkynyl group in R⁵ is optionally substituted with 1 to 2substituents independently selected from —O—C₁₋₆alkyl, —OH and phenyl;each cycloalkyl, aryl, heteroaryl and heterocycle group in R¹⁻²,R^(3a-3c), and R^(a-c) is optionally substituted with 1 to 3substituents independently selected from —C₁₋₄alkyl, —C₂₋₄alkenyl,—C₂₋₄alkynyl, cyano, halo, —O—C₁₋₄alkyl, —S—C₁₋₄alkyl, —S(O)(C₁₋₄alkyl),—S(O)₂(C₁₋₄alkyl), —NH₂, —NH(C₁₋₄alkyl) and —N(C₁₋₄alkyl)₂, wherein eachalkyl, alkenyl and alkynyl group is optionally substituted with 1 to 5fluoro substituents; and each —CH₂— group in Q is optionally substitutedwith 1 or 2 substituents independently selected from —C₁₋₂alkyl, —OH andfluoro; or a pharmaceutically acceptable salt thereof, comprising: (a)reacting a compound of formula II:

with a compound of formula III:

where L¹ represents a leaving group, to produce a compound of formulaIV:

and reacting the compound of formula IV with an organic halidecontaining an R⁵ group; or (b) reacting a compound of formula V:

with a compound of formula III; or (c) reacting a compound of formula Vwith a compound of formula VI:

where L² represents a leaving group, to produce a compound of formulaVII:

and reacting the compound of formula VII with a compound of formulaVIII:

wherein L³ represents a leaving group, and Q′, A and B are defined as:Q′ A B —CH₂— —CH₃ L³-alkylene- —CH═CH— H L³-alkenylene- —C≡C— HL³-alkynylene- —O— -L³ HO— —S— -L³ HS— —SO₂—NR^(Q1)— —SO₂—OH or —SO₂ClR^(Q1)HN— —NR^(Q1)—SO₂— —NHR^(Q1) HOO₂S— —OC(O)— —OH HO(O)C— —C(O)O——C(O)OH or —C(O)Cl HO— —NR^(Q1)C(O)— —NHR^(Q1) HO(O)C—or Cl(O)C——NR^(Q2)—C(O)—NR^(Q3)— —N═C═O H₂N— where R^(Q2) and R^(Q3) are H—NR^(Q2)—C(S)—NR^(Q3)— —N═S═O H₂N— where R^(Q2) and R^(Q3) are H —C═N—O——CHO H₂NO— —S—S— —SH HS—

and recovering the product in salt or zwitterionic form.
 2. The processof claim 1, wherein a is 0 or
 1. 3. The process of claim 1, wherein b is0.
 4. The process of claim 1, wherein R³ is —C(O)NR^(3a)R^(3b).
 5. Theprocess of claim 4, wherein R^(3a) and R^(3b) are independently selectedfrom hydrogen and —C₁₋₄alkyl.
 6. The process of claim 5, wherein R^(3a)and R^(3b) are hydrogen.
 7. The process of claim 1, wherein c is
 0. 8.The process of claim 7, wherein a and b are
 0. 9. The process of claim7, wherein a is 1 and b is
 0. 10. The process of claim 1, wherein d is 1and the double bond is absent.
 11. The process of claim 1, wherein d is2 and the double bond is present.
 12. The process of claim 1, wherein R⁵is —C₁₋₆alkyl.
 13. The process of claim 12, wherein R⁵ is —CH₃.
 14. Theprocess of claim 1, wherein —C₀₋₅alkylene-Q′-C₀₋₁alkylene- is—C₁₋₃alkylene-Q′-C₀₋₁alkylene-.
 15. The process of claim 14, wherein—C₀₋₅alkylene-Q′-C₀₋₁alkylene- is —C₃alkylene-Q′-C₀₋₁alkylene-.
 16. Theprocess of claim 1, wherein Q′ is selected from —CH₂—, —O—, —S—, —S(O)—,—C(O)—, —OC(O)—, —C(O)O—, —NR^(Q1)C(O)—, —NR^(Q2)—C(O)—NR^(Q3)-,—NR^(Q2)—C(S)—NR^(Q3)-, and —C(═N—O—R^(Q4))—.
 17. The process of claim16, wherein Q′ is —NR^(Q1)C(O)—and R^(Q1) is hydrogen.
 18. The processof claim 16, wherein Q′ is selected from —NR^(Q2)—C(O)—NR^(Q3)- and—NR^(Q2)—C(S)—NR^(Q3)-, and R^(Q2) and R^(Q3) are hydrogen.
 19. Theprocess of claim 16, wherein Q′ is —C(═N—O—R^(Q4))— and R^(Q4) is —CH₃or benzyl.
 20. The process of claim 16, wherein Q′ is selected from —O—,—S—, —C(O)—, and —OC(O)—.
 21. The process of claim 1, wherein—C₀₋₅alkylene-Q′-C₀₋₁alkylene- is selected from —(CH₂)₂, —(CH₂)₃,—(CH₂)₄, —(CH₂)₅, —(CH₂)₆, —(CH₂)₂—CH═CH—, —(CH₂)₂—C≡C—, —(CH₂)₂—O—CH₂—,—(CH₂)₃—O—, —CH₂—CH(OH)—CH₂—O—, —(CH₂)₃—O—CH₂—, —(CH₂)₃—S—,—(CH₂)₃—S(O)—, —(CH₂)₃—SO₂—, —(CH₂)₃—C(O)—, —(CH₂)₂—OC(O)—,—(CH₂)₂—C(O)O—, —CH₂—C(O)O—CH₂—, —(CH₂)₂—NR^(Q1)C(O)—,—CH₂—C(O)NR^(Q1-)CH₂, —(CH₂)₂—NR^(Q2)—C(O)—NR^(Q3)—, —CH₂—C═N—O—,—(CH₂)₂—S—S—, and —(CH₂)₃—C(═N—O—R^(Q4))—, where R^(Q1), R^(Q2) andR^(Q3) are hydrogen, and R^(Q4) is —C₁₋₄alkyl or benzyl.
 22. The processof claim 1, wherein —C₀₋₅alkylene-Q′-C₀₋₁alkylene- is —C₀₋₅alkylene-Q′-.23. The process of claim 1, wherein e is
 0. 24. The process of claim 1,wherein e is
 1. 25. The process of claim 24, wherein R⁶ is selected fromhalo, —C₁₋₄alkyl, —OH, cyano, —C(O)O—C₁₋₄alkyl, —O—C₁₋₄alkyl,—S—C₁₋₄alkyl, —NH—C(O)—C₁₋₄alkyl, —N(C₁₋₄alkyl)₂, and —N⁺(O)O.
 26. Theprocess of claim 1, wherein e is 2, one R⁶ is halo and the second R⁶ isselected from halo and —O—C₁₋₄alkyl.
 27. The process of claim 1, havingthe formula I′:

where X⁻ is an anion of a pharmaceutically acceptable acid.
 28. Theprocess of claim 27, wherein X is selected from acetate,benzenesulfonate, benzoate, bromide, butyrate, chloride,p-chlorobenzoate, citrate, diphenylacetate, formate, fluoride,o-hydroxybenzoate, p-hydroxybenzoate,1-hydroxynaphthalene-2-carboxylate, 3-hydroxynaphthalene-2-carboxylate,iodide, lactate, malate, maleate, methanesulfonate, nitrate, phosphate,propionate, succinate, sulfate, tartrate, trifluoroacetate, andtriphenylacetate.
 29. The process of claim 28, wherein X is selectedfrom bromide, iodide and trifluoroacetate.
 30. The process of claim 27,having the formula I′a:


31. The process of claim 30, having the formula I′b:


32. The process of claim 31, having the formula I′d:

wherein a and b are independently 0 or 1; each R^(l) and R² is —OR^(a),where R^(a) is hydrogen; Q is selected from: —(CH₂)₂—, —(CH₂)₃—,—(CH₂)₄—, —(CH₂)₅—, —(CH₂)₆—, —(CH₂)₂—CH═CH—, —(CH₂)₂—C≡C—,—(CH₂)₂O—CH₂—, —(CH₂)₃—O—, —CH₂—CH(OH)—CH₂—O—, —(CH₂)₃—O—CH₂—,—(CH₂)₃—S—, —(CH₂)₃—S(O)—, —(CH₂)₃—SO₂—, —(CH₂)₃—C(O)—, —(CH₂)₂—OC(O)—,—(CH₂)₂—C(O)O—, —CH₂—C(O)O—CH₂—, —(CH₂)₂—NR^(Q1)C(O)—,—CH₂—C(O)NR^(Q1)—CH₂, —(CH₂)₂—NR^(Q2)—C(O)—NR^(Q3)—, —CH₂—C═N—O—,—(CH₂)₂—S—S—, and —(CH₂)₃—C(═N—O—R^(Q4))—, where R^(Q1), R^(Q2) andR^(Q3) are hydrogen, and R^(Q4) is —C₁₋₄alkyl or benzyl; e is 0, 1 or 2;each R⁶ is independently selected from halo, —C₁₋₄alkyl,—C₀₋₄alkylene-OH, cyano, —C(O)O—C₁₋₄alkyl, —O—C₁₋₄alkyl, —S—C₁₋₄alkyl,—NH—C(O)—C₁₋₄alkyl, —N(C₁₋₄alkyl)₂, and —N⁺(O)O.
 33. The process ofclaim 32 wherein the alkyl in R⁶ is optionally substituted with 3 fluoroatoms; and one —CH₂— group in Q is optionally substituted with —OH. 34.The process of claim 32, having the formula re:

wherein a is 0 or 1; R^(l) is —OR^(a), where R^(a) is hydrogen; Q isselected from: —(CH₂)₄—, —(CH₂)₂—CH≡CH—, —(CH₂)₂—C═C—, —(CH₂)₃—O—,—(CH₂)₃—S—, —(CH₂)₃—C(O)—, and —(CH₂)₂—OC(O)—; e is 0, 1 or 2; each R⁶is independently selected from halo, —C₁₋₄alkyl, —OH, and —S—C₁₋₄alkyl.35. The process of claim 34, which is not optionally substituted. 36.The process of claim 27, having the formula I′c:


37. The process of claim 1, having the formula I″a:

where X⁻ is an anion of a pharmaceutically acceptable acid.